Methods, compositions and systems for sample deposition

ABSTRACT

Methods, compositions, systems, apparatus, and kits are provided for depositing samples onto surfaces. The samples can include one or more particles, and the surface can include one or more reaction chambers. In some embodiments, the depositing can include the use of companion particles in combination with sample particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/397,370, filed Jan. 3, 2017. U.S. patent application Ser. No.15/397,370 is a divisional of U.S. patent application Ser. No.14/515,403, filed Oct. 15, 2014. U.S. patent application Ser. No.14/515,403 is a continuation of U.S. patent application Ser. No.14/057,450, filed Oct. 18, 2013, now U.S. Pat. No. 9,150,917, issuedOct. 6, 2015. U.S. Pat. No. 9,150,917 is a continuation of PCTApplication No. PCT/US2012/034358, filed Apr. 20, 2012. PCT ApplicationNo. PCT/US2012/034358 claims benefit of U.S. Provisional Application No.61/477,358, filed Apr. 20, 2011 and claims benefit of U.S. ProvisionalApplication No. 61/546,013, filed Oct. 11, 2011 and claims benefit ofU.S. Provisional Application No. 61/585,019, filed Jan. 10, 2012. Allapplications listed in this section are incorporated herein byreference, each in its entirety.

FIELD

The present disclosure is directed towards the field of molecularbiology, in particular towards improved loading and retention of samplesonto surfaces, including nucleic acid and protein arrays.

BACKGROUND

Various techniques for analyzing biomolecules, such as polynucleotidesor proteins, rely on the deposition of an array of particles, eachattached to such biomolecules. Exemplary sequencing techniques rely onthe deposition of an array of particles including a polynucleotide orcopies thereof in an array of wells. In a particular example, theparticles or beads can be deposited within the wells to associate theparticles or beads with a particular sensor and to provide a localenvironment in which to analyze the biomolecules. In other examples, anordered array of particles are deposited on a surface and analyzedwithout the benefit of wells.

There is a challenge to load samples on a surface in an organizedmanner, such that each sample does not interfere with another sample onthe same surface. There is also a challenge to load closely spacedsamples on a surface to form an array. It is desirable to create sucharrays for nucleic acid or protein experimentation. In particular, it isdesirable to create high-density arrays suitable for sequencing ofgenomes or for sequencing of low-frequency and rare variant mutations.It would also be desirable to place nucleic acids and in particular,nucleic acids bound to a delivery particle in an organized, tightlypacked fashion, for example, to increase sequencing throughput percycle, to lower customer cost per sequenced base, to run multiplesamples in tandem, or to lower the overall amount of reagents used togenerate sequencing information from an array. However, as nucleic aciddeposition density (or nucleic acid-containing particle density) isincreased the likelihood of nucleic acid clumping and nucleic acidstacking on a surface can also increase. Additionally, less than optimalloading conditions can result in one or more nucleic acids (or nucleicacid containing particles) entering the same location on the surface,such as a well, channel, pore or groove, and interfering with downstreamdata processing. Controlled organization of nucleic acids proteins, orparticles and improved loading thereof can also simplify softwareidentification of the nucleic acids or proteins on an array.Unfortunately, when nucleic acids, particles or proteins are stacked orclumped on an array, there can be problems with interrogation for theirindividual sequence or reporter signals.

In sequencing using delivery particles coated with nucleic acids, theoverall throughput in terms of nucleic acid bases sequenced persequencing run can directly depend on the number of readable deliveryparticles coated with nucleic acids in a given interrogation area, andgenerally, the more the better. Additionally, the amount of geneticinformation processed per run is dependent on the amount of nucleic acidbases sequenced per delivery particle. When delivery particles coatedwith nucleic acids are dispensed randomly onto an array, a considerableamount of space on the array can be left open. Furthermore, somedelivery particles coated with nucleic acids can settle on the array inoverlapping fashion, settle among interstitial spaces or stacking withother particles coated with nucleic acids, which can cause difficultiesin resolving and interpreting images, signals or sequences of thenucleic acids bound to the delivery particles.

When processing an array containing particles coated with nucleic acids,it can be desirable to have the particles coated with nucleic acidspacked as densely as possible to achieve the highest possiblethroughput. For example, when sequencing particles that include nucleicacids, it can be desirable to have a single nucleic acid sequence at onelocation on the array, for example, a reaction chamber, and for thoselocations to be present at a high-density to ensure high sequencingthroughput. However, issues may arise for particles coated with nucleicacids such that the ionic field, diffraction circles or spread functionis relatively large compared to the actual size of the particles coatedwith nucleic acids. Packing the particles coated with nucleic acids at adensity such that the nucleic acid coated particles are all or mostlyall touching each other can result in un-resolvable features, whetherthese coated particles are randomly arrayed or ordered in a close pack.Thus, improved methods, compositions, systems, apparatuses and kits fordepositing samples, particularly particulate samples, onto various arraysurfaces would be desirable. Improved sequencing throughput of arrays asa result of improved sample loading would be desirable.

SUMMARY

Biomolecule enhanced particles are deposited in an array to facilitateanalysis. In an example, methods and devices for performing such methodsare utilized to deposit particles attached to polynucleotides into wellsof an array associated with an array of sensors.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure will be more fully understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 includes an illustration of an exemplary system for depositingbiomolecule-enhanced particles.

FIG. 2 includes an illustration of an exemplary well array.

FIG. 3 includes an illustration of an exemplary flow cell in cooperationwith an array.

FIG. 4 includes a block flow diagram illustrating an exemplary methodfor depositing biomolecule-enhanced particles.

FIG. 5 and FIG. 6 illustrate exemplary orientations of an array duringcentrifugation.

FIG. 7 includes a block flow diagram illustrating an exemplary methodfor depositing biomolecule-enhanced particles into an array.

FIGS. 8A-8C provide sequencing metrics associated with surfaces preparedby various embodiments of the disclosure.

FIGS. 9A-9C provide sequencing metrics associated with surfaces preparedby various embodiments of the disclosure.

FIGS. 10A-10B provide sequencing metrics associated with surfacesprepared by various embodiments of the disclosure.

FIGS. 11A and 11B provide sequencing metrics associated with surfacesprepared by various embodiments of the disclosure.

DETAILED DESCRIPTION

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits for depositing samples, forexample, particulate samples, onto one or more surfaces. In someembodiments, the disclosure relates generally to deposition of samplesonto a surface to form an array, such as a nucleic acid or proteinarray. The sample to be deposited can include one or more particles, andthe surface can include one or more reaction chambers formed therein.The particles can include one or more biomolecules attached to theparticles. The disclosure relates generally to methods and relatedcompositions, kits, systems and apparatuses for depositing a pluralityof particles into a plurality of reaction chambers. In some embodiments,the disclosed methods can include forming a particle mixture including aplurality of sample particles. The sample particles optionally have afirst average diameter. The particle mixture can optionally furtherinclude a plurality of companion particles. In some embodiments, thecompanion particles have a second average diameter greater than thefirst average diameter.

As illustrated in FIG. 1, an exemplary biomolecule-enriched particle 102includes a core 104 and biomolecules 106 attached to the core 104. Whilethe biomolecules 106 are illustrated as being disposed on a surface ofthe core 104, such biomolecules 106 can be disposed throughout the core104 and on the surface of the core 104 depending upon the porosity andpermeability of the core 104. The biomolecule-enhanced particle 102 caninclude one or more copies of the biomolecules 106. In particular, thebiomolecule-enhanced particle 102 can include at least 1000 copies ofthe biomolecules 106, such as at least 10,000 copies, at least 1,000,000copies of the biomolecules 106, or even at least 10 million copies ofthe biomolecules 106. An exemplary biomolecule 106 can include aprotein, a polynucleotide, or a combination thereof. In particular, thebiomolecule 106 can be a polynucleotide, such as DNA, RNA, derivativesthereof, or a combination thereof.

The core 104 of the biomolecule-enhanced particles 102 can be formed ofvarious inorganic or organic materials. In particular, the core 104 isan organic material, such as a polymer. For example, the polymer of thecore 104 can include a hydrophilic polymer, such as a hydrogel. Thepolymer can include silicone, polyacrylamide, polyamide, polystyrene, ora combination thereof. An inorganic material can include a glass, aceramic, or a combination thereof, such as silica, alumina, or acombination thereof.

Optionally, the biomolecule-enhanced particle 102 can be mixed insolution with a companion particle 108. In particular, the companionparticle does not include the biomolecule 106. In an example, thecompanion particle 108 has an effective diameter greater than thebiomolecule-enhance particle 102.

The system can include an array including a device layer 110 and a wallstructure 112 disposed over the device layer 110 and defining reactionchambers, such as wells 114. The biomolecule-enhanced particle 102 canbe deposited within the wells 114. In particular, the effective diameterof the biomolecule-enhanced particle 102 is not greater than theentrance of the well 114. The optional companion particle 108 can have adiameter that is greater than the entrance of the well 114. As such, thecompanion particle 108 does not fit within the well 114. In a particularexample, the companion particle 108 functions to pushbiomolecule-enhanced particles 102 into wells 114 as the companionparticle 108 traverses the surface of the well wall structure 112.Alternatively, biomolecule-enhanced particles 102 can be deposited intowells 114 without the use of companion particles. In particular, thebiomolecule-enhanced particles 102 can be driven into wells 114 usingcentrifugation, vortexing, surface tension, or other means.

As illustrated in FIG. 2, an exemplary system 200 includes a well wallstructure 202 defining an array of wells 204 disposed over oroperatively coupled to sensor pads of a sensor array. The well wallstructure 202 defines an upper surface 206. A lower surface 208associated with the well is disposed over a sensor pad of the sensorarray. The well wall structure 202 defines a sidewall 210 between theupper surface 206 and the lower surface 208. As described above,biomolecule-enhanced particles can be deposited within the wells 204defined by the well wall structure 202. Such biomolecule-enhancedparticles can be analyzed using sensors of the sensor array.

In a particular example, an array, such as a micro well array or asurface array, and associated devices can be cooperatively associatedwith a cap, defining a flow cell over the array. Further, the cap candefine fluid inlet and outlet ports, permitting flow across the array.In particular, FIG. 3 illustrates an exemplary component 300 thatincludes a cap 314 disposed over the array 306 and defining a flow celltherebetween. The cap 314 defines an inlet port 302 and an efflux port304. In an example, the efflux port 304 can be larger than the inletport 302. Fluid can be injected into the inlet port 302 and can flowacross the array 306 and out of the efflux port 304. Optionally, the cap314 defines rims 308 and 310 around the inlet port 302 and efflux port304, respectively. In use, such rims 308 and 310 can be used to form aseal with engagement structures to prevent fluid leaking onto electroniccomponents. When loading particles onto the array 306, the rims 308 and310 can define containment areas, pooling liquid samples and preventingsuch samples from flowing outside of the containment area.

Loading biomolecule-enhanced particles can include applying a portion ofa sample to the array and agitating the sample to take advantage ofdirectional forces or surface tension to motivate thebiomolecule-enhanced particles into an array, such as wells of thearray. The process can be repeated using additional portions of thesample or by reapplying a portion of the sample withdrawn from a flowcell. Optionally, the sample can include companion particles. Forexample, the sample can be prepared by coupling biomolecules to thebiomolecule-enhanced particles, optionally amplifying to make copies ofthe biomolecules on the particles, and optionally mixing thebiomolecule-enhanced particles with companion particles in a solution.In particular, the companion particles can have a diameter significantlygreater than the biomolecule-enhanced particle and optionally greaterthan an entry into the well structures within the array.

For example, as illustrated in FIG. 4, a method 400 includes applying atleast a portion of a sample to an array, as illustrated at 402. Thesample can include biomolecule-enhanced particles and optionally, caninclude companion particles. For example, a portion of the sample can bepipetted into a load port of a flow cell defined over an array.Depending on the volume of the sample and the volume of the flow cell,the entire sample can be applied to the flow cell or a portion can beapplied to the flow cell. In an example, the load port can be the inletport 302 or the efflux port 304. For example, particularly when theefflux port 304 is larger than the inlet port 302, the load port can beefflux port. When the load port is the efflux port, a sample outlet portcan be the inlet port 302. Alternatively, the outlet port can be theefflux port 304.

As illustrated at 404, the array can be centrifuged. Optionally, thearray can be held perpendicular to the plane in which the rotationoccurs during centrifugation. Alternatively, the array can be held at anangle relative to the plane within which rotation occurs duringcentrifugation. In an example, the array can face towards a centralpoint around which rotation occurs. Alternatively, the array can faceoutward and away from a central point around which rotation occurs.

For example, as illustrated in FIG. 5, an array 504 is positioned withina plane perpendicular to the plane within which rotation 502 occursduring centrifugation. As illustrated in FIG. 5, the array 504 resideswithin a plane that is parallel to the x and y-coordinates andperpendicular to the radial coordinate, r. As illustrated, thex-coordinate extends into the page and the y-coordinate extends parallelthe page. Both x and y-coordinates are orthogonal to r.

Alternatively, as illustrated in FIG. 6, an array 604 can be tiltedrelative to the plane in which rotation 602 occurs. For example, thearray 604 can be rotated around the x-axis to define an angle alpharelative to the radial direction. The angle alpha is generally greaterthan 0° and less than 180°. In a particular example, the angle alpha isnot greater than 90°, such as not greater than 75°, or even not greaterthan 65°. The angle alpha can be at least 15°, such as at least 30° oreven at least 35°.

In both of the examples of FIG. 5 and FIG. 6, a load port can bepositioned closer to a lower end of the array (504 or 604) asillustrated and an output port can be positioned closer to a higher endof the array (504 or 604) as illustrated. The reference to lower orhigher is relative to the illustrated y-axis. Alternatively, the loadport can be positioned higher than the output port. In both examples,the array can optionally face towards a center of the rotation or canalternatively face away from the center rotation. In addition, suchorientations can be changed at different steps of the method.

Following centrifugation, at least a portion of the sample can bewithdrawn from the array, as illustrated 406. In an example, the portionis withdrawn from an output port. Alternatively, the portion of thesample can be withdrawn using the same port through which the sample orportion of the sample was applied.

As illustrated at 408, a portion of the sample can be reapplied oranother portion of the sample can be applied to the array. Optionally,the other portion can be applied prior to drawing a previous portion.For example, the other portion can be applied to the load port and theprevious portion displaced by the other portion and exiting the outputport can be withdrawn.

As illustrated at 410, the process can be repeated. In particular,portions that are newly applied or reapplied to the array can becentrifuged, as described above and illustrated at 404. In an example,the process can be repeated until the sample is exhausted.Alternatively, the process can be repeated for each new portion at leastonce, such as at least twice, or even at least 3 times. Optionally, theliquid can be withdrawn from the flow cell, as illustrated at 412, oncesample has been applied and centrifuged. In a particular example, thearray can be positioned within an instrument and analysis, such assequencing, can be performed.

In an alternative example, air/liquid interfaces can be utilized tomotivate biomolecule-enhanced particles into an array, such as amicrowell array. For example, as illustrated in FIG. 7, a method 700includes creating a foam from at least a portion of the sample, asillustrated at 702. The foam can be created by aspirating gas throughthe sample. The size of bubbles can be altered using rapid pipetting orother techniques. In particular, the sample includesbiomolecule-enhanced particles. The sample may or may not includecompanion particles.

As illustrated at 704, at least a portion of the foam can be applied tothe array. For example, a portion of the foam can be applied to the loadport.

The foam can be moved or agitated, for example by vortexing the array,illustrated at 706, or using other techniques such as applying pulses ofair pressure to the foam. In an example, vortexing moves the air/liquidinterface over the microwell array, facilitating the deposition of thebiomolecule-enhanced particles into wells of the array.

Following vortexing, another portion of the foam can be applied to thearray, as illustrated at 708. The application of an additional portionof the foam can drive the previously applied portion of the foam throughan efflux port. Such used foam can be collected as a waste or can bereapplied during a later step.

As illustrated at 710, the process can be repeated and the applied foamportions can be vortexed with the array, as illustrated at 706.Optionally, once each portion of the foam has been applied to the array,the used portions of foam can be reapplied to the array or can be mixedand reapplied to the array.

Following deposition of the sample, the remaining foam can be flushedout of or withdrawn from the flow cell disposed over the array, asillustrated 712. The component including the array can be positionedwithin a device for analysis, such as a sequencing device.

In some embodiments, the method can further include contacting theparticle mixture with a surface. The surface can include at least onereaction chamber, optionally a plurality of reaction chambers. At leastsome of the reaction chambers can include an entry. Typically, the entrycan permit at least one particle to enter the reaction chamber.

In some embodiments, the reaction chamber entry (or entries), the sampleparticle and the companion particle can be sized so as to selectivelyadmit only one type of particle (sample or companion) into the reactionchamber, but not the other type. For example, in some embodiments, theaverage cross-sectional diameter of the reaction chamber entries can beless than the first average diameter of the sample particles, such thatat least some of the sample particles can enter the reaction chambersthrough the entries. In some embodiments, the average cross-sectionaldiameter of the reaction chamber entries can be greater than the secondaverage diameter of the companion particles, such that at least some ofthe companion particles cannot enter the reaction chambers through theentries and are thus substantially excluded from the reaction chambers.Alternatively, the average cross-sectional diameter of the reactionchamber entries can be greater than the first average diameter of thesample particles, such that at least some of the sample particles cannotenter the reaction chambers through the entries and are thussubstantially excluded from the reaction chambers. Further, the averagecross-sectional diameter of the reaction chamber entries can be lessthan the second average diameter of the companion particles, such thatat least some of the companion particles can enter the reaction chambersthrough the entries.

In some embodiments, the average cross sectional diameter of the entriesof the plurality of reaction chambers is greater than the first averagediameter of the sample particles. In some embodiments, the average crosssectional diameter of the entries is less than the second averagediameter.

In some embodiments, the disclosed methods can further includedepositing at least one sample particle into at least one reactionchamber of the array. Optionally, the depositing includes depositing atleast one sample particle at an identifiable position on the array.

In some embodiments, the disclosed methods can further includedepositing a sample particle of the particle mixture into a percentageof the reaction chambers of the array.

In some embodiments, the percentage of reaction chambers containing adeposited sample particle from the particle mixture can be at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, or greater.

In some embodiments, the percentage of sample particles deposited intoone or more reaction chambers on the surface can be increased relativeto the percentage of reaction chambers that are filled by a controlparticle mixture that does not include companion particles. In someembodiments, the percentage increase of reaction chambers containing adeposited sample particle from the particle mixture (as compared to thepercentage of reaction chambers containing a deposited sample particlefrom the control particle mixture) can be at least 10%, at least 20%, orat least 30%. The control particle mixture can include the same totalnumber of sample particles as the particle mixture. The control particlemixture can be identical to the particle mixture except that the controlmixture does not contain companion particles. In some embodiments, thevolume of the control particle mixture can be the same as the volume ofthe particle mixture. In some embodiments, the total number or weight ofsample particles in the control particle mixture can be the same as thetotal number or weight of sample particles in the particle mixture.

In some embodiments, the disclosed methods can further include detectingdeposition of a sample particle in a reaction chamber of the array. Thedetecting can include using a sensor, which can optionally beoperatively coupled to at least one reaction chamber of the array.

In some embodiments, the disclosed methods can further includecorrelating the deposition of at least one sample particle in thereaction chamber with improved signal to noise ratio, improvedconversion, improved key peak or improved quality of sequencing data

In some embodiments, the disclosed methods can be used for nucleic acidsequencing, such as high-throughput next generation sequencing or forprotein analysis, such as protein detection or isolation. As outlinedherein, the methods are not limited by the source of materials andtherefore include all forms of genetic and proteomic samples. Forexample, the methods can include deposition of a sample particle from aDNA, RNA, genomic DNA, cDNA, mRNA, siRNA, cDNA, lambda DNA, bacterial,viral, eukaryotic or prokaryotic source of genetic material. In someembodiments, the methods can include deposition of a sample particlefrom a full length protein, truncated protein, N-terminal protein,C-terminal protein, mutant protein, folded protein, protein fragment,purified protein and the like. Additionally, the methods are not limitedto the desired application and for example include animal, chimeric orpathogenic sequencing. The methods also include sequencing of samplessuch as environmental samples, manufacturing samples, contaminationdetection such as food supply and manufacturer's samples, and forensicsamples. In some embodiments, the methods herein can be used insemi-conductor based sequencing technology, such as Ion Torrent™ PGMSequencing.

In some embodiments, the disclosed methods can further include removinga portion of the particle mixture from the surface to enhance loading.

In some embodiments, the method can include separating one or morecompanion particles from the surface after at least one sample particleis deposited onto the surface, or into a reaction chamber formed in thesurface.

In some embodiments, the companion particles in the particle mixture canfacilitate deposition of the sample particles to the surface. Theparticle mixture can in some embodiments, comprise from about 0.5% byweight to about 90% by weight, 1% by weight to about 60% by weight, 1.5%by weight to about 40% by weight more typically from about 2% by weightto about 20% by weight, of companion particles. The companion particlesin the particle mixture can have or include an average diameter that isgreater than the average diameter of the sample particles.

In some embodiments, the sample particles deposited onto the surface, orinto one or more reaction chambers formed in or on the surface form amonolayer of sample particles.

In some embodiments, the surface can include an array, for example anarray of reaction chambers or other structures. In some embodiments, thearray can include a plurality of a reaction chambers. At least one ofthe reaction chambers can optionally be shaped so that it can include nogreater than one sample particle at a given time. In some embodiments,at least one of the reaction chambers can be a microwell.

In some embodiments, the sample particles can include one or morenucleic acid molecules attached to a delivery particle. In someembodiments, the delivery particle includes a bead.

In some embodiments, the disclosed methods for loading particles onto asurface can be useful in generating surface arrays of delivery particlesincluding nucleic acid molecules, which can be subjected to suitablemethods for sequencing one or more nucleic acid molecules attached toone or more delivery particles.

In some embodiments, the disclosure relates generally to methods (andrelated compositions, kits, systems and apparatuses) for depositing aplurality of particles into a plurality of reaction chambers. The methodincludes (a) forming a particle mixture including a plurality of sampleparticles having a first average diameter and a plurality of companionparticles having a second average diameter, and (b) contacting theparticle mixture with a surface including a plurality of reactionchambers having entries, wherein the average cross sectional diameter ofthe entries of the plurality of reaction chambers is greater than thefirst average diameter but less than the second average diameter.Optionally, the methods can further include depositing at least onesample particle into at least one reaction chamber on the array. Thedeposition of the at least one sample particle can occur at anidentifiable position on the array.

In some embodiments, the companion particles can be inert. In someembodiments, the companion particles can comprise spheres, regular orirregular shaped objects having an average diameter greater than theaverage cross sectional diameter of the one or more reaction chambers.In some embodiments, a companion particle can include at least twodifferent types of material. In some embodiments, the plurality ofcompanion particles can include at least two different subpopulations ofcompanion particles. For example, the plurality of companion particlescan include a first subpopulation of companion particles having a secondaverage diameter and a second subpopulation of companion particleshaving a third average diameter. In some embodiments, the averagediameter of at least one subpopulation of companion particles is greaterthan the average cross sectional diameter of the entries of the reactionchambers of the surface. For example, the second average diameter, thethird average diameter, or both the second and third average diameterscan be greater than the average cross sectional diameter of the entriesof the one or more reaction chambers.

According to various embodiments, the disclosure relates generally tomethods (and related compositions, kits, systems and apparatuses) forincreasing signal to noise ratio of an array, comprising: forming aparticle mixture including a plurality of sample particles having afirst average diameter and a plurality of companion particles having asecond average diameter, contacting the particle mixture with an arrayincluding a plurality of reaction chambers having entries, wherein theaverage cross section diameter of the entries of the plurality ofreaction chambers is greater than the first average diameter but lessthan the second average diameter, depositing a sample particle of theparticle mixture into a percentage of the reaction chambers, wherein areaction chamber is operable linked to a sensor, detecting deposition ofa sample particle in a reaction chamber, and correlating the depositionof at least one sample particle in the reaction chamber with improvedsignal to noise ratio, improved conversion, or improved key peak signal.

According to various embodiments, a method for improving loading densityof an array is provided that comprises: (a) forming a particle mixtureincluding a plurality of sample particles having a first averagediameter and a plurality of companion particles having a second averagediameter, and (b) contacting the particle mixture with a surface of anarray including a plurality of reaction chambers having entries, whereinthe average cross sectional diameter of the entries of the plurality ofreaction chambers is greater than the first average diameter but lessthan the second average diameter, wherein the contacting includesdepositing a sample particle of the particle mixture into a percentageof the reaction chambers on the array, and wherein the percentage isincreased relative to the percentage of reaction chambers that arefilled by a control particle mixture that does not include companionparticles.

According to various embodiments, a method for increasing signal tonoise ratio of an array is provided, which includes forming a particlemixture including a plurality of sample particles having a first averagediameter and a plurality of companion particles having a second averagediameter, contacting the particle mixture with an array including aplurality of reaction chambers having entries, wherein the average crosssection diameter of the entries of the plurality of reaction chambers isgreater than the first average diameter but less than the second averagediameter, depositing a sample particle of the particle mixture into apercentage of the reaction chambers, wherein a reaction chamber isoperable linked to a sensor, detecting deposition of a sample particlein a reaction chamber, and correlating the deposition of at least onesample particle in the reaction chamber with improved signal to noiseratio, improved conversion, or improved key peak signal.

According to various embodiments, a method of forming an ordered arrayis provided that comprises forming a particle mixture including aplurality of sample particles having a first average diameter with aplurality of companion particles having a second average diameter,contacting the particle mixture with a surface of the array having aplurality of reaction chambers having entries, wherein the average crosssectional diameter of the entries of the plurality of reaction chambersis greater than the first average diameter but less than the secondaverage diameter, and depositing at least one sample particle into atleast one reaction chamber on the array, wherein the deposition of theat least one sample particle is at an identifiable position on thearray. In some embodiments, the companion particles can be inert. Insome embodiments, the companion particles can comprise spheres, regularor irregular shaped objects having an average diameter greater than theaverage cross sectional diameter of the one or more reaction chambers.In some embodiments, a companion particle can include at least twodifferent types of material. In some embodiments, a companion particlecan include at least two different average diameters, a second averagediameter and a third average diameter, wherein the second and thirdaverage diameter are both greater than the average cross sectionaldiameter of the one or more reaction chambers.

In various embodiments, a method is provided for depositing a pluralityof particles into a plurality of reaction chambers that comprises: (a)forming a particle mixture including a plurality of sample particleshaving a first average diameter and a plurality of companion particleshaving a second average diameter, and (b) contacting the particlemixture with a surface including a plurality of reaction chambers havingentries, wherein the average cross sectional diameter of the entries ofthe plurality of reaction chambers is greater than the first averagediameter but less than the second average diameter. In some embodiments,the contacting includes depositing a sample particle of the particlemixture into a percentage of the reaction chambers. In some embodiments,the percentage of reaction chambers containing a deposited sampleparticle from the particle mixture can be at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or more. In some embodiments, thepercentage of sample particles deposited into one or more reactionchambers on the surface can be increased relative to the percentage ofreaction chambers that are filled by a control particle mixture thatdoes not include companion particles. In some embodiments, the controlparticle mixture includes the same total number of sample particles asthe particle mixture. In some embodiments, the control particle mixturecan be identical to the particle mixture except that the control mixturedoes not contain companion particles. In some embodiments, the volume ofthe control particle mixture can be the same as the volume of theparticle mixture. In some embodiments, the total number or weight ofsample particles in the control particle mixture can be the same as thetotal number or weight of sample particles in the particle mixture. Insome embodiments, the percentage increase of reaction chamberscontaining a deposited sample particle from the particle mixture (ascompared to the percentage of reaction chambers containing a depositedsample particle from the control particle mixture) can be at least 10%,at least 20%, or at least 30%.

In some embodiments, at least one of the reaction chambers of thesurface contains no greater than one sample particle. In someembodiments, when one or more sample particles are deposited in one ormore reaction chambers the sample particles can be deposited such thatone sample particle is not in direct contact with another sampleparticle on the surface. In some embodiments, one or more sampleparticles from the particle mixture deposited in the one or morereaction chambers can be separated such that the sample particles arenot touching. In some embodiments, the sample particles of the particlemixture can be deposited at a rate of one sample particle per reactionchamber. In some embodiments, the contacting can include depositing nomore than one sample particle in at least one reaction chamber on thesurface. In some embodiments, the contacting can include depositing eachof at least two sample particles into different reaction chambers.

In some embodiments, the method further comprises separating at leastone companion particle from the surface, optionally without dislodgingat least one sample particle from at least one reaction chamber. In someembodiments, the separating can include removal of at least onecompanion particle from the surface using magnetic, centrifugal,gravitational, or other forces that cause at least one of the companionparticles to be removed from the surface. In some embodiments, thesurface can be flushed with a solution such that one or more companionparticles can be separated. In some embodiments, the solution can be awashing solution. In some embodiments, the washing solution can includea detergent. In some embodiments, the separating can include removing atleast about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 99% ofthe companion particles from the surface. In some embodiments, theseparating can include repeated flushing of one or more solutions acrossthe surface until at least 60%, 70%, 80%, 90%, 95%, or more of thecompanion particles are removed from the surface. In some embodiments,the separating does not substantially affect the number of sampleparticles in the one or more reaction chambers on the surface. In someembodiments, separating companion particles from the surface dislodgesless than 15%, less than 10%, less than 5%, less than 3% of the sampleparticles deposited in the one or more reaction chambers.

In some embodiments, the method further comprises agitating the particlemixture after contacting the surface. In some embodiments, the agitatingcan include shaking, tilting, vortexing, spinning, centrifuging,concentrating, pipetting, aspirating, and the like, or other means ofassociating the sample particles with the companion particles such thatthe agitating facilities deposition of the sample particles into one ormore reaction chambers on the surface. In some embodiments, theagitating can include one or more of the above techniques or means ofassociating the sample particles with the companion particles, in anyorder, or in any combination. In some embodiments, one or more of thetechniques or means for associating the sample particles with thecompanion particles may be repeated or omitted. In some embodiments, theagitating can comprise vortexing, centrifugation and aspirating of theparticle mixture contacted with the surface. In some embodiments,agitating can comprise centrifugation, aspirating and pipetting theparticle mixture contacted with the surface. In some embodiments,agitating can comprise centrifugation, concentrating and aspirating ofthe particle mixture contacted with the surface. In some embodiments,agitating can further include removing all, or portions of the particlemixture from the surface and re-applying said portion of the particlemixture to the surface, optionally without dislodging a substantialnumber of sample particles from the reaction chambers. In someembodiments of the method, agitating can further include removing avolume of the particle mixture from the surface on one or moreoccasions, optionally without dislodging a substantial number of sampleparticles from the one or more reaction chambers. While not wishing tobe bound by the following theory, it is believed that removing a volumeof the particle mixture from the surface decreases the overall volumepresent on the surface (and in the reaction chambers) at a defined timeand consequently increases the likelihood of one or more sampleparticles being present at the air/aqueous interface on the surface. Itis contemplated that a substantial number of sample particles can bedeposited in the reaction chambers of the surface through interaction atthe aqueous/air interface.

While not wishing to be bound by the following, it is also believed thatdecreasing the overall volume of the particle mixture present on thesurface during agitating can facilitate interaction of the sampleparticles with the companion particles, and that such frequency ofinteractions, increases as the volume on the surface or in the reactionchambers decreases. It is also contemplated that in addition toincreasing the number of encounters a sample particle can have with oneor more companion particles as the particle mixture volume decreases,the probability of one or more sample particles encountering capillaryaction (for example, when the surface is a channel, pore, nanopore, wellor microwell) is increased, thereby facilitating deposition of one ormore of the sample particles in the one or more reaction chambers. Assuch, the methods outlined by the present teachings enhance depositionof sample particles from a particle mixture into one or more reactionchambers on a surface.

In some embodiments, the surface to which the one or more sampleparticles can be deposited is an array. In some embodiments, the arraycan be a nucleic acid or protein-based array. In some embodiments, thearray can be a sequencing array. In some embodiments, the array can be adetection-based array. In various embodiments of the method, the arraycan comprise a solid support, a bead array, a slide, a flowcell, amicrofluidic array, a nanofluidic array, a semiconductor-based array ora chip. In some embodiments, an array once deposited with one or moresample particles from a particle mixture can be used for nucleic acidsequencing.

In some embodiments of the method, the reaction chamber can comprise awell, channel, groove, pore, nanopore or microwell. The reaction chambercan include one or more entries to through which particles can beintroduced into the reaction chamber. Typically, if the reaction chamberincludes only one entry, then a particle or other type of sample passesthrough that entry to enter the reaction chamber. Typically, if thereaction chamber includes multiple entries, then the particle or othertype of sample passes through at least one entry of the chamber. Anysuitable surface for forming one or more reaction chambers can be used.In some embodiments, the cross sectional average diameter of the one ormore reaction chambers, or of the entries of the one or more reactionchambers, is greater than the diameter of a sample particle, but lessthan the average diameter of a companion particle. In some embodiments,the sample particles of the particle mixture can be deposited into oneor more reaction chambers on the surface. In some embodiments, areaction chamber can include a microwell, U-shaped or V-shaped well. Insome embodiments, the reaction chambers on the surface can be spherical,square, rectangular, triangular, rod-like, or hexagonal. In someembodiments, the reaction chamber can be two-sided, three-sided,four-sided, five-sided, six-sided, or more.

In various embodiments, the number of reaction chambers on the surfacecan be defined, or controlled, by the technology used to create thesurface. Any suitable method for creating or preparing reaction chamberson a surface can be used. For example, a surface containing reactionchambers can be prepared using micro-etching. In another example, asurface containing high-densities of reaction chambers can be preparedusing semiconductor technology. An exemplary high-density surfacecontaining reaction chambers prepared using semiconductor basedtechnology is an Ion Torrent™ Chip (e.g., Ion 314™, 316™ and 318™ chips,Life Technologies, CA). In the above example, the Ion 314™ chip includesabout 1.2 million reaction chambers, the Ion 316™ Chip includes about 6million reaction chambers, and the Ion 318™ chips includes about 20million reaction chambers on a single surface. In one embodiment of themethod, the number of reaction chambers includes about 1 million toabout 5 billion reaction chambers. In one embodiment of the method, thenumber of reaction chambers can be at least 1 million, at least 6million, at least 20 million, at least 50 million, at least 150 million,at least 600 million, at least 1 billion, or at least 2.5 billionreaction chambers. In some embodiments, one or more of the reactionchambers can be in contact with, operably linked to, or capacitivelycoupled to a chemical field effect transistor (chemFET) or anion-sensitive field-effect transistor (ISFET). Exemplary FET suitablefor use in the disclosed methods, as well as microwells and attendantfluidics, and methods for manufacturing them, are disclosed, forexample, in U.S. Patent Publication No. 20100301398; U.S. PatentPublication No. 20100300895; U.S. Patent Publication No. 20100300559;U.S. Patent Publication No. 20100197507, U.S. Patent Publication No.20100137143; U.S. Patent Publication No. 20090127589; and U.S. PatentPublication No. 20090026082, which are incorporated by reference intheir entireties.

In some embodiments of the method, a sample particle of the particlemixture can be a nucleic acid or protein. In some embodiments, a sampleparticle can be a fragment or portion of a protein, such as theN-terminal or C-terminal portion of one or more proteins. In someembodiments, a sample particle can be one or more nucleic acid moleculesor proteins attached to a delivery particle that has an averagecross-sectional diameter so as to be deposited into one or more reactionchambers on the surface. In some embodiments, a sample particle can beone or more nucleic acid molecules attached to a bead having an averagecross-sectional diameter that is less than the average cross-sectionaldiameter of the reaction chambers on the surface. In some embodiments,the method for depositing sample particles into a plurality of reactionchambers on the surface can be practiced on a nucleic acid molecule orprotein which can be isolated from any source, including: an organism;normal or diseased cells or tissues; body fluids; or archived tissue(e.g., tissue archived in formalin or in paraffin). Nucleic acidmolecules can be in any form, including chromosomal, genomic,organellar, methylated, cloned, amplified, DNA, cDNA, RNA, RNA/DNA orsynthesized. In some embodiments, a nucleic acid molecule can comprisenaturally occurring nucleotides, nucleotide analogs, or both. In someembodiments, a nucleic acid molecule can comprise labeled nucleic acids.Any suitable method for labeling nucleic acids may be used. For example,in some embodiments a label can include a luminescent, photoreactive orfluorescent label. In some embodiments, a label can be attached directlyto one or more nucleotides or nucleosides of the nucleic acid molecule,which in turn can be attached to a delivery particle. For example, anucleic acid molecule can be biotinylated at one end to bind with anavidin-like compound (e.g., streptavidin), acting as a deliveryparticle.

In some embodiments, the delivery particle to which a nucleic acidmolecule can be bound can include any suitable material for attachingnucleic acid molecules to the delivery particle. In some embodiments, adelivery particle can include silica, glass, coated glass, coatedpolyacrylamide, acrylamide, nylon, plastic, ceramic, porous silicon,polystyrene or a combination thereof. In some embodiments, a deliveryparticle has an average cross-sectional diameter that is less than theaverage cross sectional diameter of reaction chambers on the surface. Insome embodiments, a delivery particle can include a label, dye, bindingmoiety, magnet or detectable signal. Delivery particles can be coatedwith a carboxylic acid compound or an amine compound for attachingnucleic acid molecules. For example delivery particles can be coatedwith an avidin-like compound (e.g., streptavidin) for bindingbiotinylated nucleic acid molecules. In some embodiments, deliveryparticles can have a shape that is spherical, hemispherical,cylindrical, barrel-shaped, toroidal, rod-like, disc-like, conical,triangular, cubical, polygonal, tubular, wire-like or irregular. In someembodiments, a delivery particle can have an iron core, or comprise ahydrogel or agarose (e.g., Sepharose™). In some embodiments, a deliveryparticle can be paramagnetic. In some embodiments, a delivery particlecan have a cavitation or pore, or can include a three-dimensionalscaffold. In some embodiments, a delivery particle can be an Ion Sphere™particle.

In various embodiments of the method, sample particles can include adelivery particle and at least one nucleic acid molecule or a protein.In some embodiments, the particle mixture includes a plurality of sampleparticles and companion particles having a defined average diameter ascompared to the one or more reaction chambers of the surface. In oneembodiment, the particle mixture of the method comprises about 80% toabout 98% by weight sample particles. In some embodiments, the particlemixture comprises about 2% to about 20% by weight companion particles.In another embodiment, the particle mixture comprises less than 15%companion particles, as determined by the total number of particles inthe particle mixture. In another embodiment, the total number ofcompanion particles in the particle mixture is less than 12%, less than10%, less than 8%, less than 6%, less than 4%, or less than 2% companionparticles. In some embodiments, the average cross sectional diameter ofa sample particle can be sufficient to deposit one sample particle intoa reaction chamber on the surface. In some embodiments of the method,the average diameter of a sample particle in the particle mixture can beat least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or about 40% smaller than theaverage cross sectional diameter of the entries to the plurality ofreaction chambers on the surface. In some embodiments, the reactionchambers on the surface can include a top end and a base, wherein thetop end has a smaller average cross sectional diameter than the averagecross-sectional diameter of the base of the reaction chambers. In someembodiments, the top end of the reaction chamber can include an entry.In other embodiments, the entry can be located within the base of thereaction chamber. The entry can have an average cross sectional diameterthat is greater than the first average diameter of the sample particlesbut less than the second average diameter of the companion particles.

In various embodiments of the method, a companion particle of theparticle mixture can include a bead, and the like. In some embodiments,a companion particle can include silica, glass, coated glass, coatedpolyacrylamide, acrylamide, nylon, plastic, ceramic, porous silicon,polystyrene, latex or a combination thereof. In some embodiments, acompanion particle can be inert. In some embodiments, a companionparticle can include a label, dye, binding moiety, magnet or detectablesignal. In some embodiments, companion particles can have a shape thatis spherical, hemispherical, cylindrical, barrel-shaped, toroidal,rod-like, disc-like, conical, triangular, cubical, polygonal, tubular,wire-like or irregular. In some embodiments, a companion particle canhave an iron core, or comprise a hydrogel or agarose (e.g., Sepharose™).In some embodiments, a companion particle can be magnetic orparamagnetic. In some embodiments, a companion particle can have acavitation or pore, or can include a three-dimensional scaffold. In someembodiments, a companion particle can include coated particles, such asstreptavidin coated particles, and the like. In some embodiments, acompanion particle can include a surfactant free coating, such assurfactant free blue sulfate or surfactant free yellow green sulfate. Insome embodiments, a companion particle can include an ionic or nonioniccoating. Any suitable material for preparing a companion particle foruse in the disclosed method may be used.

In some embodiments, a companion particle can have an averagecross-sectional diameter that is greater than the average crosssectional diameter of at least some of the reaction chambers on thesurface. In some embodiments, the average diameter of a companionparticle is greater than the average diameter of a sample particle. Insome embodiments, a companion particle can comprise at least 2%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more, than theaverage diameter of a sample particle in a reaction chamber. In someembodiments, a companion particle can include one type of material. Forexample, a companion particle can include polystyrene. In someembodiments, a companion particle can include two or more types ofmaterial. For example, a companion particle can include both silica andiron. In some embodiments, a companion particle having a second averagediameter can further include a third average diameter, that is largerthan the average cross sectional diameter of the one or more reactionchambers, and is larger than the average diameter of the sampleparticles having a first average diameter.

In some embodiments, a companion particle can directly facilitatedepositing of a sample particle into a reaction chamber by physicallycontacting a sample particle and moving the sample particle to an entryof a reaction chamber, whereby the entry of the reaction chamber has anaverage cross sectional diameter that is greater than the averagediameter of the sample particle, thereby causing deposition of thesample particle into the reaction chamber. In some embodiments, thedepositing of a sample particle into a reaction chamber by a companionparticle causes formation of a monolayer of sample particles in thereaction chambers of the surface.

While not wishing to be bound by the following theory, it is believedthat a companion particle facilitates depositing of one or more sampleparticles by physically interacting and moving a sample particle on thesurface, thereby optimizing configuration of a sample particle on thesurface. It is contemplated herein that a companion particle drivesdeposition of sample particles into reaction chambers on the surface.For example, when the surface is a microwell, it is believed thatcompanion particles can physically interact with, and move a sampleparticle of the instant disclosure into a microwell, thereby depositinga sample particle into the reaction chamber. It is also contemplatedherein that a companion particle (possessing a greater average diameterthan the average diameter of a sample particle) can move a sampleparticle from interstitial spaces on the surface to a reaction chamberon the surface, thereby enhancing sample loading.

In some embodiments, a method for enhancing loading of a sample to aplurality of reaction chambers is provided comprising, applying to asurface a particle mixture including a plurality of samples particleshaving a first average diameter and a plurality of companion particleshaving a second average diameter, contacting the particle mixture withthe surface including a plurality of reaction chambers having entries,wherein the average cross sectional diameter of the entries is greaterthan the first average diameter but less than the second averagediameter, and depositing a portion of the sample particles of theparticle mixture into a percentage of the reaction chambers on thesurface. In some embodiments, after the particle mixture is contactedwith the surface, one or more of the companion particles can beseparated from the surface. In another embodiment, a portion of theparticle mixture contacted with the surface can be removed andre-applied to the surface to facilitate loading of one or more sampleparticles to the reaction chambers on the surface. In some embodiments,the method further comprises removing one or more unbound sampleparticles or one or more companion particles from the surface using awash solution. In some embodiments, a substantial amount of unboundsample particles or companion particles can be removed from the surfaceusing a wash solution. In some embodiments, the wash solution caninclude one or more salts, detergents or excipients such that the washsolution does not interfere with a sample particle in one or more of thereaction chambers. In some embodiments, the method can further includepre-loading the reaction chambers of the surface with a priming solutioncomprising an annealing buffer to enhance depositing of a sampleparticle to a reaction chamber. In some embodiments, the primingsolution can include a detergent or an alcohol. In some embodiments, thepriming solution can include isopropanol or a non-ionic detergent, ananionic detergent, a zwitterionic detergent, or a combination thereof.In some embodiments, the priming solution can include TWEEN™, TRITON™ orSDS.

In some embodiments, a sample particle in a reaction chamber can be anucleic acid molecule. In one embodiments, a sample particle in areaction chamber can be used for one or more sequencing reactions. Insome embodiments, the sequencing reaction can include single-stranded orbi-directional (paired-end) sequencing. Any suitable method ofsequencing may be used. In some embodiments, the sequencing reaction canidentify one or more mutations within the nucleic acid molecule in thereaction chamber. In some embodiments, the method can comprise applyinga sequencing polymerase to the particle mixture. In some embodiments,the method can include applying a sequencing primer to the particlemixture. In some embodiments, one or more sample particles can be boundto one or more reaction chambers, wherein the sample particles compriseone or more nucleic acid molecules. In some embodiments, the particlemixture can include sample particles from one or more DNA or nucleicacid origins. In some embodiments, one or more sample particles of theparticle mixture can include one or more tissue samples or cells from asingle source or individual. In some embodiments, the particle mixturecan include one or more tissue or cell samples from at least two sourcesor individuals. In some embodiments, the particle mixture can includenucleic acids from a multiplex sample. For example, a particle mixturecan include sample particles from at least 96, 384, 680, 1000, 3000,6000, 10000, or more different sources. In some embodiments, theparticle mixture can include a sample particle that comprises a nucleicacid molecule having a barcode sequence. In some embodiments, sampleparticles can include a plurality of nucleic acid molecules attached toa plurality of delivery particles, wherein the nucleic acid moleculescontain at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or at least99% homology. In another embodiment, sample particles can include aplurality of nucleic acid molecules attached to delivery particles,wherein the nucleic acid molecules contain substantially no homology. Asdefined herein, substantially no homology refers to nucleic acidmolecules with less than 30% homology. In some embodiments, thesequencing reactions can identify one or more mutations in one or morenucleic acids deposited in a reaction chamber. In some embodiments, themutations can include deletions, insertions, inversions or mismatches.

In some embodiments, the method further comprises contacting the surfacewith a solution of control nucleic acid particles that can include knownnucleic acid sequences. In some embodiments, the control nucleic acidparticles can be included in the particle mixture and contacted with thesurface. In some embodiments, one or more of the control nucleic acidparticles can be deposited in one or more of the reaction chambers andused for sequencing. In some embodiments, the control nucleic acidparticles can be sequenced and used to obtain a baseline or backgroundsequence from one or more of the reaction chambers. In some embodiments,the control nucleic acid particles can include a reference nucleic acidsample such that the reference sample can be compared to the sampleparticles in the reaction chambers. In some embodiments, the referencesample can be compared to the nucleic acid sequences obtained from thesample particles in the reaction chambers to identify the sequence ofthe sample particles or identify the present of one or mutations in thesample particles as compared to the reference sample. In someembodiments, the reference sample is a genomic or amplicon sample. Insome embodiments, the reference sample is a biological, clinical,agricultural, manufacturing or environmental sample. In someembodiments, the reference sample is a bacterial, viral, fungal, plant,animal or chimeric nucleic acid sample. Any suitable reference samplecan be used to obtain a background or reference nucleic acid sequence,using sequencing methods known to those skilled in the art.

In some embodiments, the method further comprises applying a foamingsolution to the surface. In some embodiments, the foaming solution canbe applied prior to, concurrently with, or after contacting the particlemixture with the surface. In some embodiments, the foaming solution caninclude polyethylene gycol or polysorbate. In various embodiments of themethod, the foaming solution can include one or more detergents. In someembodiments, the detergent can be a non-ionic detergent, an anionicdetergent, a zwitterionic detergent, or a combination thereof. In someexemplary embodiments, the detergent can include TWEEN™, TRITON™, BRIJ™,or SDS.

According to various embodiments, a method of improving sample loadingis provided that comprises forming a particle mixture including aplurality of sample particles having a first average diameter and aplurality of companion particles having a second average diameter,contacting the particle mixture with a surface including a plurality ofreaction chambers having entries, wherein the average cross sectionaldiameter of the entries of the plurality of reaction chambers is greaterthan the first average diameter but less than the second averagediameter, and wherein the contacting includes depositing a sampleparticle of the particle mixture into a percentage of reaction chambers,wherein the percentage is increased relative to the percentage ofreaction chambers that are filled by a control particle mixture thatdoes not include companion particles.

In some embodiments, the method further comprises separating at leastone companion particle from the surface. In another embodiment, themethod further comprises priming a reaction chamber of the surface witha priming solution including an annealing buffer so as to facilitatedeposition of a sample particle into a reaction chamber. In someembodiments, the reaction chambers of the surface are contained in anarray. In some embodiments, the array can be a flow cell, slide,nanofluidic array, or a bead array and the reaction chamber can be amicrowell, groove or nanopore. In some embodiments of the method, thenumber of reaction chambers on the surface can include about 1 millionto about 5 billion reaction chambers. In some embodiments, a sampleparticle of the particle mixture can include a nucleic acid molecule. Insome embodiments, a nucleic acid molecule in a reaction chamber can beused in one or more sequencing reactions. In some embodiments, asequencing reaction can include analyzing the nucleic acid molecule ofthe sample particle to a reference nucleic acid sequence. In someembodiments, the particle mixture can include one or more controlnucleic acid particles. In some embodiments, a control nucleic acidparticle can act as an internal control or a positive control todemonstrate that the sequencing process has been completed or issuccessful. In some embodiments of the method, the method furtherincludes a sequencing polymerase or a sequencing primer can be includedin the particle mixture. In some embodiments, a sequencing primer caninclude a sequence that is complementary to one or more nucleic acidsequences on the one or more sample particles in the reaction chambers.In some embodiments, a sequencing primer can hybridize to a sampleparticle under appropriate hybridizing conditions. In some embodiments,a sequencing polymerase can include any suitable polymerase sufficientto bind to, or associate with, a nucleic acid molecule attached to adelivery particle in a reaction chamber, so as to promote or facilitateextension of the sequencing primer hybridized to the nucleic acidmolecule. In some embodiments, extension of a sequencing primer caninclude incorporation of a nucleotide and concommant release of ahydrogen atom. In some embodiments, the release of a hydrogen ion duringnucleotide incorporation can be associated with the type of nucleotideincorporated and therefore can determine the sequence of the extendedsequencing primer.

Provided herein are kits for deposition of a plurality of sampleparticles into a plurality of reaction chambers on a surface. In someembodiments, the kits relate generally to nucleic acid and protein basedarrays. In some embodiments, the kits relate to sequencing of one ormore nucleic acids deposited into a reaction chamber of the surface. Insome embodiments, kits include any reagent that can be used to contact aparticle mixture including a plurality of sample particles and aplurality of companion particles with a surface. In some embodiments,the kits can include any one or more of the following components: acompanion particle, a surface, which can optionally include a pluralityof reaction chambers, buffers; cations; one or more primers; one or moreenzymes; one or more nucleotides; reagents for nucleic acidpurification; or reagents for nucleic acid amplification. In someembodiments, kits include any combination of: polymerase(s); ligase(s);endonuclease(s); kinase(s); phosphatase(s); or nuclease(s).

The present teachings will be more fully understood with reference tothe following Examples that are intended to illustrate, not limit, thepresent teachings.

EXAMPLES Example 1

The following discloses a non-limiting example of a method to prepare anIon Torrent 314™ Chip (Life Technologies, Part No. 4462923) and methodfor enhancing nucleic acid loading on the Ion Torrent 314™ Chip forsequencing using an Ion Torrent PGM™ Sequencer (Life Technologies, PartNo. 4462917).

Chip Preparation

1. A new Ion Torrent 314™ Chip was obtained and labeled appropriately toidentify the experiment.2. A Chip check and calibration on the PGM™ sequencer was performedessentially according to the protocols provided in the Ion SequencingKit User Guide v2.0 (Life Technologies, Part No. 4468997), herebyincorporated by reference in its entirety, using Ion PGM™ Supplies Kit(Life Technologies, Part No. 4468996), Ion Sequencing Reagents Kit (LifeTechnologies, Part No. 4468995) and Ion PGM™ Reagents Kit (LifeTechnologies, Part No, 4468994).3. The Chip was removed from the PGM™ sequencer and washed with 50 μL100% Isopropanol (2-propanol) and then washed with 3× of 50 μL AnnealingBuffer (sold as a component of the Ion PGM™ Reagents Kit, (LifeTechnologies, Part No. 4468994). Here, each washing step entailed a 2minute spin on a microcentrifuge at 14,000 rpm.4. A final rinse of the Chip was performed using 50 μL of 50% Annealingbuffer.

It is preferred that when the Chip is not in the PGM™ Sequencer andclamped in position, that a dummy chip is loaded into the PGM™ sequencerto prevent air pockets from forming due to back flow in the squid lines.

The Proceeding steps required the preparation of nucleic-acid loadedbeads (here, Ion Sphere Particles loaded with DNA). Procedures toperform and generate DNA-loaded ISPs can be practiced essentiallyaccording to the protocols provided in the Ion Xpress™ Template Kit UserGuide v2.0 (Life Technologies, Part No. 4469004), hereby incorporated byreference in its entirety, using the Ion Xpress™ Template Kit (LifeTechnologies, Part No. 4469001), hereby incorporated by reference in itsentirety.

Enhanced Nucleic Acid Loading

1. 10 million DNA-loaded ISPs or half a plate of ISPs prepared using theIon Xpress™ Template Kit were transferred into a 200 μL PCR tube and 1μL of Ion Sphere™ Test Fragments (sold as a component of the Ion ControlMaterial Kit, Life Technologies, Part No. 4466465) were added. If theDNA-loaded ISPs are in excess of 50 μL, Annealing Buffer (sold as acomponent of the Ion PGM™ Reagents Kit, (Life Technologies, Part No.4468994) was added up to ˜150 μL and centrifuged once to concentrate.The supernatant was removed to a volume of 25 μL.2. The DNA-loaded ISPs were washed by filling the sample tube with 150μL Annealing Buffer and gently mixed by stirring with the tip of apipette.3. The sample containing the DNA-loaded ISPs of step 2 were centrifugedfor 2 minutes at a minimum of 15000 rcf. The supernatant was discarded,except for a final volume of 5 μL. The 5 μL volume was mixed by repeatedpipetting.4. 6 μL of Sequencing Primer (sold as a component of the Ion SequencingReagents Kit, (Life Technologies, Part No. 4468995) was added to the 5μL volume of step 3. If the volume of step 3 is less than 5 μL, thefinal volume is adjusted by adding Annealing Buffer to a final volume of11 μL. If adjustment is required, mix the sample well by pipetting.5. The sample of step 4 was run on the following hybridization programon a thermocycler (for example a “QuickHyb Program”) with the followingtemperature profile:

-   -   95° C., for 2 minutes, followed by 37° C., for 2 minutes.        6. 1 μL of Sequencing Polymerase (sold as a component of the Ion        Sequencing Reagents Kit, (Life Technologies, Part No. 4468995)        was added to the sample after performing the QuickHyb Program        and the sample was mixed and incubated at room temperature for 5        minutes.

During Incubation:

7. A solution (30 μl) containing companion particles (1.5 million)(here,SOLiD EZ Bead 6 um polystyrene beads) was transferred into a 1.5 ml PCRtube, to which 500 ul of 50% Annealing Buffer was added. The solutionwas vortexed and centrifuged for 2 minutes at a minimum of 15000 rcf.The supernatant was completely removed carefully so as not to disturbthe particle pellet.8. Once the incubation period was complete, the mixture of step 6 (˜12μL) was added to the tube containing companion particles (step 7). Thesample was mixed using a pipette at least 10 times.9. The sample was then sonicated for 10 seconds. Droplets may bedeposited on the interior wall of the tube. If this occurs, collect theliquid into one pool by a brief (1-2 seconds) spin in a picocentrifugeand then briefly mix with the tip of a pipette.10. With the Chip on a flat surface, 6 μL of the sample (from step 9)was applied to the loading port (large port) of the Chip, dialing downthe pipette to gently and slowly deposit the sample into the Chip. Agood speed is about 1 μL every second. Residual liquid was removed fromthe outlet port.11. The Chip was then centrifuged at room temperature for 1 minute.12. Using a pipette, 5 μL of the applied sample was removed from theChip (at the outlet port) and re-applied to the Chip (via the loadingport), 5 times.13. The Chip was then centrifuged at room temperature for 30-60 seconds.14. Steps 12 and 13 were repeated once.15. All liquid was removed gently from the Chip.16. Steps 10-15 were repeated using the second half of the sample fromstep 9.17. The Chip was then washed 4 times with 50 ul of 50% Annealing Bufferto remove companion particles or unbound ISPs before inserting Chip intoPGM™ Sequencer.18. A PGM™ run was performed essentially according to the protocolsprovided in the Ion Sequencing Kit User Guide v2.0 (Life Technologies,Part No. 4468997), under the heading “Ion 314™ Chip—Begin theExperiment”.

Example 2

The following discloses a non-limiting example of a method to prepare anIon Torrent 316™ Chip (Life Technologies, Part No. 4469496) and methodfor enhancing nucleic acid loading on the Ion Torrent 316™ Chip using anIon Torrent PGM™ Sequencer (Life Technologies, Part No. 4462917).

Chip Preparation

1. A new Ion Torrent 316™ Chip was obtained and labeled appropriately toidentify the experiment.2. A Chip check and calibration on the PGM™ sequencer was performedessentially according to the protocols provided in the Ion SequencingKit User Guide v2.0 (Life Technologies, Part No. 4468997), herebyincorporated by reference in its entirety, using Ion PGM™ Supplies Kit(Life Technologies, Part No. 4468996), Ion Sequencing Reagents Kit (LifeTechnologies, Part No. 4468995) and Ion PGM™ Reagents Kit (LifeTechnologies, Part No, 4468994).3. The Chip was removed from the PGM™ sequencer and washed with 100 μL100% Isopropanol (2-propanol) and then washed with 3× of 100 μLAnnealing Buffer (sold as a component of the Ion PGM™ Reagents Kit,(Life Technologies, Part No. 4468994). Here, each washing step entaileda 2 minute spin on a microcentrifuge at 14,000 rpm.4. A final rinse of the Chip was performed using 100 μL of 50% Annealingbuffer.

It is preferred that when the Chip is not in the PGM™ Sequencer andclamped in position, that a dummy chip is loaded into the PGM™ sequencerto prevent air pockets from forming due to back flow in the squid lines.

The Proceeding steps required the preparation of nucleic-acid loadedbeads (here, Ion Sphere Particles loaded with DNA). Procedures toperform and generate DNA-loaded ISPs can be practiced essentiallyaccording to the protocols provided in the Ion Xpress™ Template Kit UserGuide v2.0 (Life Technologies, Part No. 4469004), hereby incorporated byreference in its entirety, using the Ion Xpress™ Template Kit (LifeTechnologies, Part No. 4469001), hereby incorporated by reference in itsentirety.

Enhanced Nucleic Acid Loading

1. 20 million DNA-loaded ISPs or one full plate of ISPs prepared usingthe Ion Xpress™ Template Kit were transferred into a 200 μL PCR tube and2 μL Ion Sphere™ Test Fragments from the Ion Control Material Kit (LifeTechnologies, Part No. 4466465) were added. If the DNA-loaded ISPs arein excess of 50 μL, Annealing Buffer (sold as a component of the IonPGM™ Reagents Kit, (Life Technologies, Part No. 4468994) was added up to˜150 μL and centrifuged once to concentrate. The supernatant was removedto a volume of 25 μL.2. The DNA-loaded ISPs were washed by filling the sample tube with 150μL Annealing Buffer and gently mixed using the tip of pipette.3. The sample containing the DNA-loaded ISPs of step 2 were centrifugedfor 2 minutes at a minimum of 15000 rcf. The supernatant was discarded,except for a final volume of 15 μL. The 15 μL volume was mixed byrepeated pipetting.4. 12 μL of Sequencing Primer (sold as a component of the Ion SequencingReagents Kit, (Life Technologies, Part No. 4468995) was added to the 15μL volume of step 3. If the volume of step 3 is less than 15 μL, thefinal volume is adjusted by adding Annealing Buffer to a final volume of27 μL. If adjustment is required, mix the sample well by pipetting.5. The sample of step 4 was run on the following hybridization programon a thermocycler (for example a “QuickHyb Program”) with the followingtemperature profile:

-   -   95° C., for 2 minutes, followed by 37° C., for 2 minutes.        6. 3 μL of Sequencing Polymerase (sold as a component of the Ion        Sequencing Reagents Kit, (Life Technologies, Part No. 4468995)        was added to the sample after performing the QuickHyb Program        and the sample was mixed and incubated at room temperature for 5        minutes.

During Incubation:

7. A solution (70 μL) containing companion particles (3.5 million)(here,SOLiD EZ Bead 6 um polystyrene beads) was transferred into a 1.5 ml PCRtube, to which 500 μL of 50% Annealing Buffer was added. The solutionwas vortexed and centrifuged for 2 minutes at a minimum of 15000 rcf.The supernatant was removed carefully so as not to disturb particlepellet and resuspended in 30 μL 50% Annealing buffer.8. Once the incubation period was complete, the mixture of step 6 (˜30μL) was added to the tube containing 30 μL of companion particles (step7).9. The sample was sonicated for 10 seconds. Droplets may be deposited onthe interior wall of the tube. If this occurs, collect the liquid intoone pool by a brief (1-2 seconds) spin in a picocentrifuge and thenbriefly mix the sample using the tip of a pipette.10. With the Chip on a flat surface, 30 μL of the sample (from step 9)was applied to the loading port (large port) of the Chip, dialing downthe pipette to gently and slowly deposit the sample into the Chip. Agood speed is about 1 μL every second. Residual liquid was removed fromthe outlet.11. The Chip was then centrifuged at room temperature for 1 minute.12. Using a pipette, 25 μL of the applied sample was removed from theChip (via the outlet port) and re-applied to the Chip (via the loadingport), 5 times.13. The Chip was then centrifuged at room temperature for 30-60 seconds.14. Steps 12 and 13 were repeated once.15. All liquid was removed gently from the Chip.16. Steps 10-15 were repeated using the second half of the sample fromstep 9.17. The Chip was then washed 4 times with 50 μL of 50% Annealing Bufferto remove companion particles and unbound ISPs before inserting Chipinto PGM™ Sequencer.18. A PGM™ run was performed essentially according to the protocolsprovided in the Ion Sequencing Kit User Guide v2.0 (Life Technologies,Part No. 4468997), under the heading “Ion 316™ Chip—Begin theExperiment”.

Example 3

The following discloses a non-limiting example of a method to prepare anIon Torrent 318™ Chip (Life Technologies) and method for enhancingsample loading on the Ion Torrent 318™ Chip using an Ion Torrent PGM™Sequencer (Life Technologies, Part No. 4462917).

Chip Preparation

1. A new Ion Torrent 318™ Chip was obtained and labeled appropriately toidentify the experiment.2. A Chip check and calibration on the PGM™ sequencer was performedessentially according to the protocols provided in the Ion SequencingKit User Guide v2.0 (Life Technologies, Part No. 4468997), herebyincorporated by reference in its entirety, using Ion PGM™ Supplies Kit(Life Technologies, Part No. 4468996), Ion Sequencing Reagents Kit (LifeTechnologies, Part No. 4468995) and Ion PGM™ Reagents Kit (LifeTechnologies, Part No, 4468994).3. The Chip was removed from the PGM™ sequencer and washed with 100 μL100% Isopropanol (2-propanol) and then washed with 3× of 100 μLAnnealing Buffer (sold as a component of the Ion PGM™ Reagents Kit,(Life Technologies, Part No. 4468994). Here, each washing step entaileda 2 minute spin on a microcentrifuge at 14,000 rpm.4. A final rinse of the Chip was performed using 100 μL of 50% Annealingbuffer.

It is preferred that when the Chip is not in the PGM™ Sequencer andclamped in position, that a dummy chip is loaded into the PGM™ sequencerto prevent air pockets from forming due to back flow in the squid lines.

The Proceeding steps required the preparation of nucleic-acid loadedbeads (here, Ion Sphere Particles loaded with DNA). Procedures toperform and generate DNA-loaded ISPs can be practiced essentiallyaccording to the protocols provided in the Ion Xpress™ Template Kit UserGuide v2.0 (Life Technologies, Part No. 4469004), hereby incorporated byreference in its entirety, using the Ion Xpress™ Template Kit (LifeTechnologies, Part No. 4469001), hereby incorporated by reference in itsentirety.

Enhanced Nucleic Acid Loading (ISPs)

1. 30 million DNA-loaded ISPs or one full plate of ISPs prepared usingthe Ion Xpress™ Template Kit were transferred into a 200 μL PCR tube and2 μL Ion Sphere™ Test Fragments from the Ion Control Material Kit (LifeTechnologies, Part No. 4466465) were added. If the DNA-loaded ISPs arein excess of 50 μL, Annealing Buffer (sold as a component of the IonPGM™ Reagents Kit, (Life Technologies, Part No. 4468994) was added up to˜150 μL and centrifuged once to concentrate. The supernatant was removedto a volume of 25 μl.2. The DNA-loaded ISPs were washed by filling the sample tube with 150μL Annealing Buffer and gently mixed with the tip of a pipette.3. The sample containing the DNA-loaded ISPs of step 2 were centrifugedfor 2 minutes at a minimum of 15000 rcf. The supernatant was discarded,except for a final volume of 15 μL. The 15 μL volume was mixed byrepeated pipetting.4. 12 μL of Sequencing Primer (sold as a component of the Ion SequencingReagents Kit, (Life Technologies, Part No. 4468995) was added to the 15μL volume of step 3. If the volume of step 3 is less than 15 μL, thefinal volume is adjusted by adding Annealing Buffer to a final volume of27 μL. If adjustment is required, mix the sample well by pipetting.5. The sample of step 4 was run on the following hybridization programon a thermocycler (for example a “QuickHyb Program”) with the followingtemperature profile:

-   -   95° C., for 2 minutes, followed by 37° C., for 2 minutes.        6. 3 μL of Sequencing Polymerase (sold as a component of the Ion        Sequencing Reagents Kit, (Life Technologies, Part No. 4468995)        was added to the sample after performing the QuickHyb Program        and the sample was mixed and incubated at room temperature for 5        minutes.

During Incubation:

7. A solution (100 μL) containing companion particles (5 million)(here,SOLiD EZ Bead 6 um polystyrene beads) was transferred into a 1.5 ml PCRtube, to which 500 μL of 50% Annealing Buffer was added. The solutionwas vortexed and centrifuged for 2 minutes at a minimum of 15000 rcf.The supernatant was removed carefully so as not to disturb particlepellet and resuspended in 30 μL 50% Annealing buffer.8. Once the incubation period was complete, the mixture of step 6 (˜30μL) was added to the tube containing 30 μL of companion particles (step7).9. The sample was sonicated for 10 seconds. Droplets may be deposited onthe interior wall of the tube. If this occurs, collect the liquid intoone pool by a brief (1-2 seconds) spin in a picocentrifuge and thenbriefly mixed with the tip of a pipette.10. With the Chip on a flat surface, 30 μL of the sample (from step 9)was applied to the loading port (large port) of the Chip, dialing downthe pipette to gently and slowly deposit the sample into the Chip. Agood speed is about 1 μL every second. Residual liquid was removed fromthe outlet.11. The Chip was then centrifuged at room temperature for 1 minute.12. Using a pipette, 25 μL of the applied sample was removed from theChip and re-applied to the Chip (via the loading port), 5 times.13. The Chip was then centrifuged at room temperature for 30-60 seconds.14. Steps 12 and 13 were repeated once.15. All liquid was removed gently from the Chip.16. Steps 10-15 were repeated using the second half of the sample fromstep 9.17. The Chip was then washed 4 times with 50 μl of 50% Annealing Bufferto remove companion particles and unbound ISPs before inserting Chipinto PGM™ Sequencer.18. A PGM™ run was performed essentially according to the protocolsprovided in the Ion Sequencing Kit User Guide v2.0 (Life Technologies,Part No. 4468997).

Example 4

The following discloses another non-limiting example of a method toprepare an Ion Torrent 316™ Chip (Life Technologies, Part No. 4469496)and method for enhancing sample loading on the Ion Torrent 316™ Chipusing an Ion Torrent PGM™ Sequencer (Life Technologies, Part No.4462917).

Chip Preparation

1. A new Ion Torrent 316™ Chip was obtained and labeled appropriately toidentify the experiment.2. A Chip check and calibration on the PGM™ sequencer was performedessentially according to the protocols provided in the Ion SequencingKit User Guide v2.0 (Life Technologies, Part No. 4468997), herebyincorporated by reference in its entirety, using Ion PGM™ Supplies Kit(Life Technologies, Part No. 4468996), Ion Sequencing Reagents Kit (LifeTechnologies, Part No. 4468995) and Ion PGM™ Reagents Kit (LifeTechnologies, Part No, 4468994).3. The Chip was removed from the PGM™ sequencer and washed with 100 μL100% Isopropanol (2-propanol) and then washed with 3× of 100 μLAnnealing Buffer (sold as a component of the Ion PGM™ Reagents Kit,(Life Technologies, Part No. 4468994). Here, each washing step entaileda 2 minute spin on a microcentrifuge at 14,000 rpm.4. All residual fluid was removed from the Chip. For example, by tiltingthe chip such that any remaining fluid flows to the outlet port where itwas removed using a pipette.

It is preferred that when the Chip is not in the PGM™ Sequencer andclamped in position, that a dummy chip is loaded into the PGM™ sequencerto prevent air pockets from forming due to back flow in the squid lines.

The proceeding steps required the preparation of nucleic-acid loadedbeads (here, Ion Sphere Particles loaded with DNA). Procedures toperform and generate DNA-loaded ISPs can be practiced essentiallyaccording to the protocols provided in the Ion Xpress™ Template Kit UserGuide v2.0 (Life Technologies, Part No. 4469004), hereby incorporated byreference in its entirety, using the Ion Xpress™ Template Kit (LifeTechnologies, Part No. 4469001), hereby incorporated by reference in itsentirety.

Enhanced Nucleic Acid Loading (ISPs)

1. 20 million DNA-loaded ISPs or one full plate of ISPs prepared usingthe Ion Xpress™ Template Kit were transferred into a 200 μL PCR tube and24 Ion Sphere™ Test Fragments from the Ion Control Material Kit (LifeTechnologies, Part No. 4466465) were added. If the DNA-loaded ISPs arein excess of 50 μL, Annealing Buffer (sold as a component of the IonPGM™ Reagents Kit, (Life Technologies, Part No. 4468994) was added up to˜150 μL and centrifuged once to concentrate. The supernatant was removedto a volume of 25 μl.2. The DNA-loaded ISPs were washed by filling the sample tube with 150μL Annealing Buffer and gently mixed with the tip of a pipette.3. The sample containing the DNA-loaded ISPs of step 2 were centrifugedfor 2 minutes at a minimum of 15000 rcf. The supernatant was discarded,except for a final volume of 15 μL. The 15 μL volume was mixed byrepeated pipetting.4. 12 μL of Sequencing Primer (sold as a component of the Ion SequencingReagents Kit, (Life Technologies, Part No. 4468995) was added to the 15μL volume of step 3. If the volume of step 3 is less than 15 μL, thefinal volume is adjusted by adding Annealing Buffer to a final volume of27 μL. If adjustment is required, mix the sample well by pipetting.5. The sample of step 4 was run on the following hybridization programon a thermocycler (for example a “QuickHyb Program”) with the followingtemperature profile:95° C., for 2 minutes, followed by 37° C., for 2 minutes.6. 3 μL of Sequencing Polymerase (sold as a component of the IonSequencing Reagents Kit, (Life Technologies, Part No. 4468995) was addedto the sample after performing the QuickHyb Program and the sample wasmixed and incubated at room temperature for 5 minutes.

During Incubation:

7. A solution (70 μL) containing companion particles (3.5 million)(here,SOLiD EZ Bead 6 um polystyrene beads) was transferred into a 1.5 ml PCRtube, to which 500 μL of annealing buffer was added. The solution wasvortexed and centrifuged for 2 minutes at a minimum of 15000 rcf. Thesupernatant was removed carefully so as not to disturb particle pelletand resuspended in 30 μL Annealing buffer.8. Once the incubation period was complete, the mixture of step 6 (˜30μL) was added to the tube containing 30 μL of companion particles (step7).9. The sample was sonicated for 10 seconds. Droplets may be deposited onthe interior wall of the tube. If this occurs, collect the liquid intoone pool by a brief (1-2 seconds) spin in a picocentrifuge and thenbriefly mixed using the tip of a pipette.10. With the Chip on a flat surface, 30 μL of the sample (from step 9)was applied to the loading port (large port) of the Chip, dialing downthe pipette to gently and slowly deposit the sample into the Chip. Agood speed is about 1 μL every second. Residual liquid was removed fromthe outlet.11. Small covers were placed over each of the Chip ports.12. The Chip was then centrifuged at room temperature for 1 minute.13. The Chip was then subjected to four rounds of vortexing andcentrifugation under the following conditions: Vortex in IKA MS3 Shakerat 2000 rpm for 20 seconds, followed by centrifugation for 20 seconds.14. The covers were removed from the ports of the Chip and all liquidwas removed from the Chip.15. Steps 10-14 were repeated with the second half of the sample fromstep 9.16. The Chip was then washed 4 times with 50 μL of 50% Annealing Bufferto remove companion particles or unbound ISPs before inserting Chip intoPGM™ Sequencer.17. A PGM™ run was performed essentially according to the protocolsprovided in the Ion Sequencing Kit User Guide v2.0 (Life Technologies,Part No. 4468997).

Example 5

The following discloses another non-limiting example of a method toprepare an Ion Torrent 316™ Chip (Life Technologies, Part No. 4469496)and method for enhancing sample loading on the Ion Torrent 316™ Chipusing an Ion Torrent PGM™ Sequencer (Life Technologies, Part No.4462917).

Chip Preparation

1. A new Ion Torrent 316™ Chip was obtained and labeled appropriately toidentify the experiment.2. A Chip check and calibration on the PGM™ sequencer was performedessentially according to the protocols provided in the Ion SequencingKit User Guide v2.0 (Life Technologies, Part No. 4468997), herebyincorporated by reference in its entirety, using Ion PGM™ Supplies Kit(Life Technologies, Part No. 4468996), Ion Sequencing Reagents Kit (LifeTechnologies, Part No. 4468995) and Ion PGM™ Reagents Kit (LifeTechnologies, Part No, 4468994).3. The Chip was removed from the PGM™ sequencer and washed with 100 μL100% Isopropanol (2-propanol) and then washed with 3× of 100 μLAnnealing Buffer (sold as a component of the Ion PGM™ Reagents Kit,(Life Technologies, Part No. 4468994). Here, each washing step entaileda 2 minute spin on a microcentrifuge at 14,000 rpm.4. All residual fluid was removed from the Chip. For example, by tiltingthe chip such that any remaining fluid flows to the outlet port where itwas removed using a pipette.

It is preferred that when the Chip is not in the PGM™ Sequencer andclamped in position, that a dummy chip is loaded into the PGM™ sequencerto prevent air pockets from forming due to back flow in the squid lines.

The proceeding steps required the preparation of nucleic-acid loadedbeads (here, Ion Sphere Particles loaded with DNA). Procedures toperform and generate DNA-loaded ISPs can be practiced essentiallyaccording to the protocols provided in the Ion Xpress™ Template Kit UserGuide v2.0 (Life Technologies, Part No. 4469004), hereby incorporated byreference in its entirety, using the Ion Xpress™ Template Kit (LifeTechnologies, Part No. 4469001), hereby incorporated by reference in itsentirety.

Enhanced Nucleic Acid Loading (ISPs)

1. 20 million DNA-loaded ISPs or a full plate of ISPs prepared using theIon Xpress™ Template Kit were transferred into a 200 μL PCR tube and 24Ion Sphere™ Test Fragments from the Ion Control Material Kit (LifeTechnologies, Part No. 4466465) were added. If the DNA-loaded ISPs arein excess of 50 μL, Annealing Buffer (sold as a component of the IonPGM™ Reagents Kit, (Life Technologies, Part No. 4468994) was added up to˜150 μL and centrifuged once to concentrate. The supernatant was removedto a volume of 25 μl.2. The DNA-loaded ISPs were washed by filling the sample tube with 150μL Annealing Buffer and gently mixed with the tip of a pipette.3. The sample containing the DNA-loaded ISPs of step 2 were centrifugedfor 2 minutes at a minimum of 15000 rcf. The supernatant was discarded,except for a final volume of 15 μL. The 15 μL volume was mixed byrepeated pipetting4. 12 μL of Sequencing Primer (sold as a component of the Ion SequencingReagents Kit, (Life Technologies, Part No. 4468995) was added to the 15μL volume of step 3. If the volume of step 3 is less than 15 μL, thefinal volume is adjusted by adding Annealing Buffer to a final volume of27 μL. If adjustment is required, mix the sample well by pipetting.5. The sample of step 4 was run on the following hybridization programon a thermocycler (for example a “QuickHyb Program”) with the followingtemperature profile:

-   -   95° C., for 2 minutes, followed by 37° C., for 2 minutes.        6. 3 μL of Sequencing Polymerase (sold as a component of the Ion        Sequencing Reagents Kit, (Life Technologies, Part No. 4468995)        was added to the sample after performing the QuickHyb Program        and the sample was mixed and incubated at room temperature for 5        minutes.

During Incubation:

7. A solution (70 μL) containing companion particles (3.5 million)(here,SOLiD EZ Bead 6 um polystyrene beads) was transferred into a 1.5 ml PCRtube, to which 500 μL of Annealing buffer was added. The solution wasvortexed and centrifuged for 2 minutes at a minimum of 15000 rcf. Thesupernatant was removed carefully so as not to disturb the particlepellet and resuspended in 30 μL Annealing buffer.8. Once the incubation period was complete, the mixture of step 6 (˜30μL) was added to the tube containing 30 μL of companion particles (step7).9. The sample was sonicated for 10 seconds. Droplets may be deposited onthe interior wall of the tube. If this occurs, collect the liquid intoone pool by a brief (1-2 seconds) spin in a picocentrifuge and thenbriefly mixed using the tip of a pipette.10. With the Chip on a flat surface, 30 μL of the sample (from step 9)was applied to the loading port (large port) of the Chip, dialing downthe pipette to gently and slowly deposit the sample into the Chip. Agood speed is about 1 μL every second. Residual liquid was removed fromthe outlet.11. Small covers were placed over each of the Chip ports.12. The Chip was then centrifuged at room temperature for 1 minute.13. The Chip was then subjected to two rounds of vortexing andcentrifugation under the following conditions: Vortex in IKA MS3 Shakerat 2000 rpm for 20 seconds, followed by centrifugation for 20 seconds.14. The covers were removed from the ports of the Chip and all residualfluid was removed from the Chip.15. Steps 10-14 were repeated with the second half of the sample fromstep 9.16. The Chip was then washed 4 times with 50 μL of 50% Annealing Bufferto remove companion particles and unbound ISPs before inserting Chipinto PGM™ Sequencer.17. A PGM™ run was performed essentially according to the protocolsprovided in the Ion Sequencing Kit User Guide v2.0 (Life Technologies,Part No. 4468997).

Example 6

FIGS. 8A-8C show data obtained using methods of the present disclosure.

FIG. 8A shows the number of 100 base pair read lengths as rated byquality scores of Q17.

FIG. 8B shows the number of 200 base pair read lengths as rated byquality scores of Q17.

FIG. 8C shows the total number of bases from a single run having aquality score of Q17.

Each of the above criteria was tested using six experimental conditions[1]-[6]. Experiments [1] and [2] refer to testing conditions in theabsence of companion particles. Experiments [3]-[6] refer to testingconditions in the presence of companion particles. Experiment [3] TOBB,[4] PTOBB (Example 2, herein), [5] Short Chimera (Example 5, herein) and[6] Chimera (Example 4, herein) refer to loading of a nucleic acid arrayusing the methods disclosed herein. Specifically, experiments [3]-[6]involve a different attaching step(s) and include one or more of thetechniques disclosed herein to attach nucleic acid bound particles tothe array.

Experiments [3]-[6] that included companion particles, were observed toresult in an increase in the number of reads (FIGS. 8A and 8B) or totalthroughput (FIG. 8C) as compared to non-companion particle based arrays.Additionally, the mean length of each read with a Q17 score was found tobe longer for experiments including companion particles as compared tonon-companion particle counterparts (FIG. 9B). Furthermore, the signalto noise ratio (FIG. 9C) improved when using companion particles on anarray, as compared to non-companion particle arrays. In each figure, thenumbers in parenthesis corresponds to the standard deviation of the fiveindependent experiments (n=5).

FIGS. 10A and 10B provide data from arrays prepared using companionparticles (experiments [3]-[6]), as compared to arrays prepared in theabsence of companion particles (experiments [1] and [2]). As can be seenfrom the data, experiments [3]-[6] were generally observed to generatehigher ratios, less standard derivation (indicative of more readable andgreater read lengths of nucleic acids bound to the array), as comparedto non-companion particle based arrays.

Example 7

The following discloses another non-limiting example of a method forenhancing sample loading on the Ion Torrent 316™ Chip using an IonTorrent PGM™ Sequencer (Life Technologies, Part No. 4462917).

The proceeding steps required the preparation of nucleic-acid loadedbeads (here, Ion Sphere Particles loaded with DNA). Procedures toperform and generate DNA-loaded ISPs can be practiced essentiallyaccording to the protocols provided in the Ion Xpress™ Template Kit UserGuide v2.0 (Life Technologies, Part No. 4469004), hereby incorporated byreference in its entirety, using the Ion Xpress™ Template Kit (LifeTechnologies, Part No. 4469001), hereby incorporated by reference in itsentirety.

Enhanced Nucleic Acid Loading (ISPs)

1. 30 million DNA-loaded ISPs or a full plate of ISPs prepared using theIon Xpress™ Template Kit were transferred into a 200 μL PCR tube and 2μL Ion Sphere™ Test Fragments from the Ion Control Material Kit (LifeTechnologies, Part No. 4466465) were added. If the DNA-loaded ISPs arein excess of 50 μL, Annealing Buffer (sold as a component of the IonPGM™ Reagents Kit, (Life Technologies, Part No. 4468994) was added up to˜150 μL and centrifuged once to concentrate. The supernatant was removedto a volume of 12 μl.2. 12 μL of Sequencing Primer (sold as a component of the Ion SequencingReagents Kit, (Life Technologies, Part No. 4468995) was added to the 12μL volume of step 1.4. The sample of step 3 was run on the following hybridization programon a thermocycler (for example a “QuickHyb Program”) with the followingtemperature profile:

-   -   95° C., for 2 minutes, followed by 37° C., for 2 minutes.        5. 6 μL of foaming solution (10% Triton-X100 in annealing        buffer) was added to the sample of step 4 and mixed.        6. The sample of step 5 was mixed to create foam as follows: air        bubbles were injected into the sample of step 5 using a pipette        tip, with repeated pipetting (up and down) for about 15 seconds        to create a homogenous consistency of small air bubbles. If        useful to obtain a consistency of small air bubbles, this step        was repeated.        7. 40 μL of the foam created in step 6, was applied to the Chip        and centrifuged at 3000 rpm for 2-3 minutes. Any excess solution        in the outlet port was discarded.        8. Another 30 μL of the foam created in step 6, was applied to        the Chip and centrifuged at 3000 rpm for 2-3 minutes. Any excess        solution in the outlet port was discarded.        9. Steps 7 and 8 were repeated with the remaining foam created        in step 6.        10. After all the foam created in step 6 was applied to the        Chip, the chip was flushed with 100 μL of 100% isopropanol and        twice flushed with 100 μL of annealing buffer.        11. 3 μL of Sequencing Polymerase (sold as a component of the        Ion Sequencing Reagents Kit, (Life Technologies, Part        No. 4468995) was added to 30 μL of annealing buffer and mixed        slowly.        11. With the Chip on a flat surface, the sample (from step 11)        was applied to the loading port (large port) of the Chip,        dialing down the pipette to gently and slowly deposit the sample        into the Chip.        12. The Chip was then incubated at room temperature for 5        minutes.        13. The Chip was then inserted into the PGM™ Sequencer and a        PGM™ run was performed essentially according to the protocols        provided in the Ion Sequencing Kit User Guide v2.0 (Life        Technologies, Part No. 4468997).

Example 8

The following discloses a non-limiting example of a method to prepare anIon Torrent 314™ Chip (Life Technologies, Part No. 4462923) and methodfor enhancing nucleic acid loading on the Ion Torrent 314™ Chip forsequencing using an Ion Torrent PGM™ Sequencer (Life Technologies, PartNo. 4462917).

Chip Preparation

1. A new Ion Torrent 314™ Chip was obtained and labeled appropriately toidentify the experiment.2. A chip check and calibration on the PGM™ sequencer was performedessentially according to the protocols provided in the Ion SequencingKit User Guide v2.0 (Life Technologies, Part No. 4468997), herebyincorporated by reference in its entirety, using Ion PGM™ Supplies Kit(Life Technologies, Part No. 4468996), Ion Sequencing Reagents Kit (LifeTechnologies, Part No. 4468995) and Ion PGM™ Reagents Kit (LifeTechnologies, Part No, 4468994).3. The Chip was removed from the PGM™ sequencer and washed with 100 μL100% Isopropanol (2-propanol) and then washed with 3× of 100 μLAnnealing Buffer (sold as a component of the Ion PGM™ Reagents Kit,(Life Technologies, Part No. 4468994). Here, each washing step entaileda 2 minute spin on a microcentrifuge at 14,000 rpm (16873 rcf).

The proceeding steps required the preparation of nucleic-acid loadedbeads (here, Ion Sphere Particles (ISPs) loaded with DNA). Procedures toperform and generate DNA-loaded ISPs can be practiced essentiallyaccording to the protocols provided in the Ion Xpress™ Template Kit UserGuide v2.0 (Life Technologies, Part No. 4469004), hereby incorporated byreference in its entirety, using the Ion Xpress™ Template Kit (LifeTechnologies, Part No. 4469001), hereby incorporated by reference in itsentirety.

Enhanced Nucleic Acid Loading (ISPs)

1. 10 million DNA-loaded ISPs or a half plate of ISPs prepared using theIon Xpress™ Template Kit were transferred into a 200 μL PCR tube and 2μL Ion Sphere™ Test Fragments from the Ion Control Material Kit (LifeTechnologies, Part No. 4466465) were added. If the DNA-loaded ISPs arein excess of 50 μL, Annealing Buffer (sold as a component of the IonPGM™ Reagents Kit, (Life Technologies, Part No. 4468994) was added up to˜150 μL and centrifuged once to concentrate. The supernatant was removedto a volume of 25 μl.2. The DNA-loaded ISPs were washed by filling the sample tube with 150μL Annealing Buffer and gently mixed with the tip of a pipette.3. The sample containing the DNA-loaded ISPs of step 2 were centrifugedfor 2 minutes at a minimum of 15000 rcf. The supernatant was discarded,except for a final volume of 3 μL. The 3 μL volume was mixed by repeatedpipetting.4. 3 μL of Sequencing Primer (sold as a component of the Ion SequencingReagents Kit, (Life Technologies, Part No. 4468995) was added to the 3μL volume of step 3. If the final volume of step 4 is less than 6 μL,the volume was adjusted by adding Annealing Buffer to a final volume of6 μL. If adjustment is required, mix the sample well by pipetting.5. The sample of step 4 was run on the following hybridization programon a thermocycler (for example a “QuickHyb Program”) with the followingtemperature profile:

-   -   95° C. for 2 minutes; followed by 37° C. for 2 minutes.        6. 1 μL of Sequencing Polymerase (sold as a component of the Ion        Sequencing Reagents Kit, (Life Technologies, Part No. 4468995)        was added to the sample after performing the QuickHyb Program        and the sample was mixed and incubated at room temperature for 5        minutes.        7. The loading protocol was then bifurcated based on the        presence of companion particles in the loading protocol. If        companion particles were to be used, the following steps were        performed during incubation of the sample from step 6.        8. A solution (30 μL) containing companion particles (3.5        million)(here, SOLiD EZ Bead 6 um polystyrene beads) was        transferred into a 200 μl PCR tube, to which 150 μL of 50%        Annealing Buffer was added. The solution was vortexed and        centrifuged for 2 minutes at a minimum of 15000 rcf. The        supernatant was removed carefully so not to disturb the particle        pellet.        9. Once the incubation period was complete, the incubated        mixture of step 6 (˜7 μL) was added to the tube containing        companion particles (step 8).        10. The sample was sonicated for 10 seconds. Droplets may        deposit on the interior wall of the tube. If this occurs,        collect the liquid into one pool by a brief (1-2 seconds) spin        in a centrifuge and then briefly mix using the tip of a pipette.        11. With the Chip on a flat surface, 7 μL of the sample (from        step 10) was applied to the loading port (large port) of the        Chip, dialing down the pipette to gently and slowly deposit the        sample into the Chip. A good speed is about 1 μL every second.        Residual liquid was removed from the outlet.        12. The Chip was then centrifuged at room temperature for 1        minute.        13. 4 μl of the loaded sample volume was removed and re-loaded        (re-applied) to the chip port for a minimum of five cycles,        while leaving some liquid in the chip to minimize air bubbles.        14. Steps 12 and 13 were repeated once.        15. All residual fluid was removed from the Chip.        16. The Chip was then washed 4 times with 50 μL of 50% Annealing        Buffer to remove companion particles and unbound ISPs before        inserting Chip into PGM™ Sequencer.        17. A PGM™ run was performed essentially according to the        protocols provided in the Ion Sequencing Kit User Guide v2.0        (Life Technologies, Part No. 4468997), incorporated herein by        reference in their entirety.

Example 9

The following discloses a non-limiting example of a method to prepare anIon Torrent 316™ Chip (Life Technologies, Part No. 4462923) and methodfor enhancing nucleic acid loading on the Ion Torrent 316™ Chip forsequencing using an Ion Torrent PGM™ Sequencer (Life Technologies, PartNo. 4462917).

Chip Preparation

1. A new Ion Torrent 316™ Chip was obtained and labeled appropriately toidentify the experiment.2. A chip check and calibration on the PGM™ sequencer was performedessentially according to the protocols provided in the Ion SequencingKit User Guide v2.0 (Life Technologies, Part No. 4468997), herebyincorporated by reference in its entirety, using Ion PGM™ Supplies Kit(Life Technologies, Part No. 4468996), Ion Sequencing Reagents Kit (LifeTechnologies, Part No. 4468995) and Ion PGM™ Reagents Kit (LifeTechnologies, Part No, 4468994).3. The Chip was removed from the PGM™ sequencer and washed with 100 μL100% Isopropanol (2-propanol) and then washed with 3× of 100 μLAnnealing Buffer (sold as a component of the Ion PGM™ Reagents Kit,(Life Technologies, Part No. 4468994). Here, each washing step entaileda 2 minute spin on a microcentrifuge at 14,000 rpm (16873 rcf).

The proceeding steps required the preparation of nucleic-acid loadedbeads (here, Ion Sphere Particles (ISPs) loaded with DNA). Procedures toperform and generate DNA-loaded ISPs can be practiced essentiallyaccording to the protocols provided in the Ion Xpress™ Template Kit UserGuide v2.0 (Life Technologies, Part No. 4469004), hereby incorporated byreference in its entirety, using the Ion Xpress™ Template Kit (LifeTechnologies, Part No. 4469001), hereby incorporated by reference in itsentirety.

Enhanced Nucleic Acid Loading (ISPs)

1. 20 million DNA-loaded ISPs or a full plate of ISPs prepared using theIon Xpress™ Template Kit were transferred into a 200 μL PCR tube and 3μL Ion Sphere™ Test Fragments from the Ion Control Material Kit (LifeTechnologies, Part No. 4466465) were added. If the DNA-loaded ISPs arein excess of 50 μL, Annealing Buffer (sold as a component of the IonPGM™ Reagents Kit, (Life Technologies, Part No. 4468994) was added up to˜150 μL and centrifuged once to concentrate. The supernatant was removedto a volume of 25 μL.2. The DNA-loaded ISPs were washed by filling the sample tube with 150μL Annealing Buffer and gently mixed with the tip of a pipette.3. The sample containing the DNA-loaded ISPs of step 2 were centrifugedfor 2 minutes at a minimum of 15000 rcf. The supernatant was discarded,except for a final volume of 15 μL. The 15 μL volume was mixed byrepeated pipetting.4. 12 μL of Sequencing Primer (sold as a component of the Ion SequencingReagents Kit, (Life Technologies, Part No. 4468995) was added to the 15μL volume of step 3. If the final volume of step 4 is less than 27 μLthe volume was adjusted by adding Annealing Buffer to a final volume of27 μL. If adjustment is required, mix the sample well by pipetting.5. The sample of step 4 was run on the following hybridization programon a thermocycler (for example a “QuickHyb Program”) with the followingtemperature profile:

-   -   95° C. for 2 minutes; followed by 37° C. for 2 minutes.        6. 3 μL of Sequencing Polymerase (sold as a component of the Ion        Sequencing Reagents Kit, (Life Technologies, Part No. 4468995)        was added to the sample after performing the QuickHyb Program        and the sample was mixed and incubated at room temperature for 5        minutes. The final volume should be about 30 μL.        7. The loading protocol was then bifurcated based on the        presence of companion particles in the loading protocol. If        companion particles were to be used, the following steps were        performed during incubation of the sample from step 6.        8. A solution (70 μL) containing companion particles (5        million)(here, SOLiD EZ Bead 6 um polystyrene beads) was        transferred into a 1.5 ml PCR tube, to which 500 μL of 50%        Annealing Buffer was added. The solution was vortexed and        centrifuged for 2 minutes at a minimum of 15000 rcf. The        supernatant was removed carefully so not to disturb the particle        pellet.        9. Once the incubation period was complete, the incubated        mixture of step 6 (˜30 μL) was added to the tube containing        companion particles (step 8).        10. The sample was sonicated for 10 seconds. Droplets may        deposit on the interior wall of the tube. If this occurs,        collect the liquid into one pool by a brief (1-2 seconds) spin        in a centrifuge and then briefly mix using the tip of a pipette.        11. With the Chip on a flat surface, 30 μL of the sample (from        step 10) was applied to the loading port (large port) of the        Chip, dialing down the pipette to gently and slowly deposit the        sample into the Chip. A good speed is about 1 μL every second.        Residual liquid was removed from the outlet.        12. The Chip was then centrifuged at room temperature for 1        minute.        13. 25 μl of the loaded sample volume was removed and re-loaded        (re-applied) to the chip port for a minimum of five cycles,        while leaving some liquid in the chip to minimize air bubbles.        14. Steps 12 and 13 were repeated once.        15. All residual fluid was removed from the Chip. In this        instance, the chip was held at a 45-degree angle, to gently        remove as much residual liquid as possible from the port of the        chip. If residual liquid remains, the following steps were        performed:        a) Hold the chip with the ports facing outward from your palm        with the loading port being lower. b) Pipette as much liquid as        possible from the loading port.        c) With a flick of the wrist, draw the remaining liquid to the        loading port and remove        d) Repeat steps a)-c) until as much liquid is removed as        possible.        16. The Chip was then washed 4 times with 50 μL of 50% Annealing        Buffer to remove companion particles and unbound ISPs before        inserting Chip into PGM™ Sequencer.        17. A PGM™ run was performed essentially according to the        protocols provided in the Ion Sequencing Kit User Guide v2.0        (Life Technologies, Part No. 4468997), incorporated herein by        reference in their entirety.

Example 10

The following discloses a non-limiting example of a method to prepare anIon Torrent 318™ Chip (Life Technologies) and method for enhancingnucleic acid loading on the Ion Torrent 318™ Chip for sequencing usingan Ion Torrent PGM™ Sequencer (Life Technologies, Part No. 4462917).

Chip Preparation

1. A new Ion Torrent 318™ Chip was obtained and labeled appropriately toidentify the experiment.2. A chip check and calibration on the PGM™ sequencer was performedessentially according to the protocols provided in the Ion SequencingKit User Guide v2.0 (Life Technologies, Part No. 4468997), herebyincorporated by reference in its entirety, using Ion PGM™ Supplies Kit(Life Technologies, Part No. 4468996), Ion Sequencing Reagents Kit (LifeTechnologies, Part No. 4468995) and Ion PGM™ Reagents Kit (LifeTechnologies, Part No, 4468994).3. The Chip was removed from the PGM™ sequencer and washed with 100 μL100% Isopropanol (2-propanol) and then washed with 3× of 100 μLAnnealing Buffer (sold as a component of the Ion PGM™ Reagents Kit,(Life Technologies, Part No. 4468994). Here, each washing step entaileda 2 minute spin on a microcentrifuge at 14,000 rpm (16873 rcf).

The proceeding steps required the preparation of nucleic-acid loadedbeads (here, Ion Sphere Particles (ISPs) loaded with DNA). Procedures toperform and generate DNA-loaded ISPs can be practiced essentiallyaccording to the protocols provided in the Ion Xpress™ Template Kit UserGuide v2.0 (Life Technologies, Part No. 4469004), hereby incorporated byreference in its entirety, using the Ion Xpress™ Template Kit (LifeTechnologies, Part No. 4469001), hereby incorporated by reference in itsentirety.

Enhanced Nucleic Acid Loading (ISPs)

1. 30 million DNA-loaded ISPs or a full plate of ISPs prepared using theIon Xpress™ Template Kit were transferred into a 200 μL PCR tube and 5μL Ion Sphere™ Test Fragments from the Ion Control Material Kit (LifeTechnologies, Part No. 4466465) were added. If the DNA-loaded ISPs arein excess of 50 μL, Annealing Buffer (sold as a component of the IonPGM™ Reagents Kit, (Life Technologies, Part No. 4468994) was added up to˜150 μL and centrifuged once to concentrate. The supernatant was removedto a volume of 25 μl.2. The DNA-loaded ISPs were washed by filling the sample tube with 150μL Annealing Buffer and gently mixed with the tip of a pipette.3. The sample containing the DNA-loaded ISPs of step 2 were centrifugedfor 2 minutes at a minimum of 15000 rcf. The supernatant was discarded,except for a final volume of 15 μL. The 15 μL volume was mixed byrepeated pipetting. 4. 12 μL of Sequencing Primer (as a component of theIon Sequencing Reagents Kit, (Life Technologies, Part No. 4468995) wasadded to the 15 μL volume of step 3. If the final volume of step 4 isless than 27 μL, the volume was adjusted by adding Annealing Buffer to afinal volume of 27 μL. If adjustment is required, mix the sample well bypipetting.5. The sample of step 4 was run on the following hybridization programon a thermocycler (for example a “QuickHyb Program”) with the followingtemperature profile:

-   -   95° C. for 2 minutes; followed by 37° C. for 2 minutes.        6. 3 μL of Sequencing Polymerase (sold as a component of the Ion        Sequencing Reagents Kit, (Life Technologies, Part No. 4468995)        was added to the sample after performing the QuickHyb Program        and the sample was mixed and incubated at room temperature for 5        minutes. The final volume should be about 30 μL.        7. The loading protocol was then bifurcated based on the        presence of companion particles in the loading protocol. If        companion particles were to be used, the following steps were        performed during incubation of the sample from step 6.        8. A solution (100 μL) containing companion particles (7.5        million)(here, SOLiD EZ Bead 6 um polystyrene beads) was        transferred into a 1.5 ml PCR tube, to which 500 μL of 50%        Annealing Buffer was added. The solution was vortexed and        centrifuged for 2 minutes at a minimum of 15000 rcf. The        supernatant was removed carefully so not to disturb the particle        pellet.        9. Once the incubation period was complete, the incubated        mixture of step 6 (˜30 μL) was added to the tube containing        companion particles (step 8).        10. The sample was sonicated for 10 seconds. Droplets may        deposit on the interior wall of the tube. If this occurs,        collect the liquid into one pool by a brief (1-2 seconds) spin        in a centrifuge and then briefly mix using the tip of a pipette.        11. With the Chip on a flat surface, 30 μL of the sample (from        step 10) was applied to the loading port (large port) of the        Chip, dialing down the pipette to gently and slowly deposit the        sample into the Chip. A good speed is about 1 μL every second.        Residual liquid was removed from the outlet.        12. The Chip was then centrifuged at room temperature for 1        minute.        13. 25 μL of the loaded sample volume was removed and slowly        re-loaded (re-applied) to the chip port for a minimum of five        cycles, while leaving some liquid in the chip to minimize air        bubbles.        14. Steps 12 and 13 were repeated once.        15. All residual fluid was removed from the Chip. In this        instance, the chip was held at a 45-degree angle, to gently        remove as much residual liquid as possible from the port of the        chip. If residual liquid remains, the following steps were        performed:        a) Hold the chip with the ports facing outward from your palm        with the loading port being lower. b) Pipette as much liquid as        possible from the loading port.        c) With a flick of the wrist, draw the remaining liquid to the        loading port and remove        d) Repeat steps a)-c) until as much liquid is removed as        possible.        16. The Chip was then washed 4 times with 50 μL of 50% Annealing        Buffer to remove companion particles and unbound ISPs before        inserting Chip into PGM™ Sequencer.        17. A PGM™ run was performed essentially according to the        protocols provided in the Ion Sequencing Kit User Guide v2.0        (Life Technologies, Part No. 4468997), incorporated herein by        reference in their entirety.

Example 11

The following discloses a non-limiting example of a method to prepare anIon Torrent 316™ Chip (Life Technologies, Part No. 4462923) and methodfor enhancing nucleic acid loading on the Ion Torrent 316™ Chip forsequencing using an Ion Torrent PGM™ Sequencer (Life Technologies, PartNo. 4462917).

Chip Preparation

1. A new Ion Torrent 316™ Chip was obtained and labeled appropriately toidentify the experiment.2. A chip check and calibration on the PGM™ sequencer was performedessentially according to the protocols provided in the Ion SequencingKit User Guide v2.0 (Life Technologies, Part No. 4468997), herebyincorporated by reference in its entirety, using Ion PGM™ Supplies Kit(Life Technologies, Part No. 4468996), Ion Sequencing Reagents Kit (LifeTechnologies, Part No. 4468995) and Ion PGM™ Reagents Kit (LifeTechnologies, Part No, 4468994).3. The Chip was removed from the PGM™ sequencer and washed with 100 μL100% Isopropanol (2-propanol) and then washed with 3× of 100 μLAnnealing Buffer (sold as a component of the Ion PGM™ Reagents Kit,(Life Technologies, Part No. 4468994). Here, each washing step entaileda 2 minute spin on a microcentrifuge at 14,000 rpm (16873 rcf).

The proceeding steps required the preparation of nucleic-acid loadedbeads (here, Ion Sphere Particles (ISPs) loaded with DNA). Procedures toperform and generate DNA-loaded ISPs can be practiced essentiallyaccording to the protocols provided in the Ion Xpress™ Template Kit UserGuide v2.0 (Life Technologies, Part No. 4469004), hereby incorporated byreference in its entirety, using the Ion Xpress™ Template Kit (LifeTechnologies, Part No. 4469001), hereby incorporated by reference in itsentirety.

Enhanced Nucleic Acid Loading (ISPs)

1. 20 million DNA-loaded ISPs or a full plate of ISPs prepared using theIon Xpress™ Template Kit were transferred into a 200 μL PCR tube and 3μL Ion Sphere™ Test Fragments from the Ion Control Material Kit (LifeTechnologies, Part No. 4466465) were added. If the DNA-loaded ISPs arein excess of 50 μL, Annealing Buffer (sold as a component of the IonPGM™ Reagents Kit, (Life Technologies, Part No. 4468994) was added up to˜150 μL and centrifuged once to concentrate. The supernatant was removedto a volume of 25 μl.2. The DNA-loaded ISPs were washed by filling the sample tube with 150μL Annealing Buffer and gently mixed with the tip of a pipette.3. The sample containing the DNA-loaded ISPs of step 2 were centrifugedfor 2 minutes at a minimum of 15000 rcf. The supernatant was discarded,except for a final volume of 15 μL. The 15 μL volume was mixed byrepeated pipetting.4. 12 μL of Sequencing Primer (sold as a component of the Ion SequencingReagents Kit, (Life Technologies, Part No. 4468995) was added to the 15μL volume of step 3. If the final volume of step 4 is less than 27 μL,the volume was adjusted by adding Annealing Buffer to a final volume of27 μL. If adjustment is required, mix the sample well by pipetting.5. The sample of step 4 was run on the following hybridization programon a thermocycler (for example a “QuickHyb Program”) with the followingtemperature profile:

-   -   95° C. for 2 minutes; followed by 37° C. for 2 minutes.        6. 3 μL of Sequencing Polymerase (sold as a component of the Ion        Sequencing Reagents Kit, (Life Technologies, Part No. 4468995)        was added to the sample after performing the QuickHyb Program        and the sample was mixed and incubated at room temperature for 5        minutes. The final volume should be about 30 μL.        7. The sample was sonicated for 10 seconds. Droplets may deposit        on the interior wall of the tube. If this occurs, collect the        liquid into one pool by a brief (1-2 seconds) spin in a        centrifuge and then briefly mix using the tip of a pipette.        8. With the Chip on a flat surface, 30 μL of the sample (from        step 6) was applied to the loading port (large port) of the        Chip, dialing down the pipette to gently and slowly deposit the        sample into the Chip. A good speed is about 1 μL every second.        Residual liquid was removed from the outlet.        9. The Chip was then centrifuged at room temperature for 1        minute.        10. 20 μL of the loaded sample volume was removed and slowly        re-loaded (re-applied) to the chip port for a minimum of five        cycles, while leaving some liquid in the chip to minimize air        bubbles.        11. The Chip was then centrifuged at room temperature for 1        minute.        12. Steps 10 and 11 were repeated once.        13. All residual fluid was removed from the Chip. In this        instance, the chip was held at a 45-degree angle, to gently        remove as much residual liquid as possible from the port of the        chip. If residual liquid remains, the following steps were        performed:        a) Hold the chip with the ports facing outward from your palm        with the loading port being lower. b) Pipette as much liquid as        possible from the loading port.        c) With a flick of the wrist, draw the remaining liquid to the        loading port and remove        d) Repeat steps a)-c) until as much liquid is removed as        possible.        14. The Chip was then washed 1 time with 50 μL of 50% Annealing        Buffer before inserting Chip into PGM™ Sequencer.        15. A PGM™ run was performed essentially according to the        protocols provided in the Ion Sequencing Kit User Guide v2.0        (Life Technologies, Part No. 4468997), incorporated herein by        reference in their entirety.

Example 12

The following discloses a non-limiting example of a method to prepare anIon Torrent 316™ Chip (Life Technologies, Part No. 4462923) and methodfor enhancing nucleic acid loading on the Ion Torrent 316™ Chip forsequencing using an Ion Torrent PGM™ Sequencer (Life Technologies, PartNo. 4462917).

Chip Preparation

1. A new Ion Torrent 316™ Chip was obtained and labeled appropriately toidentify the experiment.2. A chip check and calibration on the PGM™ sequencer was performedessentially according to the protocols provided in the Ion SequencingKit User Guide v2.0 (Life Technologies, Part No. 4468997), herebyincorporated by reference in its entirety, using Ion PGM™ Supplies Kit(Life Technologies, Part No. 4468996), Ion Sequencing Reagents Kit (LifeTechnologies, Part No. 4468995) and Ion PGM™ Reagents Kit (LifeTechnologies, Part No, 4468994).3. The Chip was removed from the PGM™ sequencer and washed with 100 μL100% Isopropanol (2-propanol) and then washed with 3× of 100 μLAnnealing Buffer (sold as a component of the Ion PGM™ Reagents Kit,(Life Technologies, Part No. 4468994). Here, each washing step entaileda 2 minute spin on a microcentrifuge at 14,000 rpm (16873 rcf).

The proceeding steps required the preparation of nucleic-acid loadedbeads (here, Ion Sphere Particles (ISPs) loaded with DNA). Procedures toperform and generate DNA-loaded ISPs can be practiced essentiallyaccording to the protocols provided in the Ion Xpress™ Template Kit UserGuide v2.0 (Life Technologies, Part No. 4469004), hereby incorporated byreference in its entirety, using the Ion Xpress™ Template Kit (LifeTechnologies, Part No. 4469001), hereby incorporated by reference in itsentirety.

Enhanced Nucleic Acid Loading (ISPs

1. 20 million DNA-loaded ISPs or a full plate of ISPs prepared using theIon Xpress™ Template Kit were transferred into a 200 μL PCR tube and 3μL Ion Sphere™ Test Fragments from the Ion Control Material Kit (LifeTechnologies, Part No. 4466465) were added. If the DNA-loaded ISPs arein excess of 50 μL, Annealing Buffer (sold as a component of the IonPGM™ Reagents Kit, (Life Technologies, Part No. 4468994) was added up to˜150 μL and centrifuged once to concentrate. The supernatant was removedto a volume of 25 μl.2. The DNA-loaded ISPs were washed by filling the sample tube with 150μL Annealing Buffer and gently mixed with the tip of a pipette.3. The sample containing the DNA-loaded ISPs of step 2 were centrifugedfor 2 minutes at a minimum of 15000 rcf. The supernatant was discarded,except for a final volume of 15 μL. The 15 μL volume was mixed byrepeated pipetting.4. 12 μL of Sequencing Primer (sold as a component of the Ion SequencingReagents Kit, (Life Technologies, Part No. 4468995) was added to the 15μL volume of step 3. If the final volume of step 4 is less than 27 μL,the volume was adjusted by adding Annealing Buffer to a final volume of27 μL. If adjustment is required, mix the sample well by pipetting.5. The sample of step 4 was run on the following hybridization programon a thermocycler (for example a “QuickHyb Program”) with the followingtemperature profile: 95° C. for 2 minutes; followed by 37° C. for 2minutes.6. 3 μL of Sequencing Polymerase (sold as a component of the IonSequencing Reagents Kit, (Life Technologies, Part No. 4468995) was addedto the sample after performing the QuickHyb Program and the sample wasmixed and incubated at room temperature for 5 minutes. The final volumeshould be about 30 μL.7. The sample was sonicated for 10 seconds. Droplets may deposit on theinterior wall of the tube. If this occurs, collect the liquid into onepool by a brief (1-2 seconds) spin in a centrifuge and then briefly mixusing the tip of a pipette.8. With the Chip on a flat surface, 30 μL of the sample (from step 6)was applied to the loading port (large port) of the Chip, dialing downthe pipette to gently and slowly deposit the sample into the Chip. Agood speed is about 14 every second. Residual liquid was removed fromthe outlet.9. The Chip was then centrifuged at room temperature for 1 minute.10. 25 μl of the loaded sample volume was removed and slowly re-loaded(re-applied) to the chip port for a minimum of five cycles, whileleaving some liquid in the chip to minimize air bubbles.11. The Chip was then centrifuged at room temperature for 1 minute.12. Steps 10 and 11 were repeated once.12. After the final centrifugation, check to ensure no large bubbles aretrapped on the chip. If present, the chip was flushed with 100 μl of 50%Annealing Buffer before inserting Chip into PGM™ Sequencer.13. A PGM™ run was performed essentially according to the protocolsprovided in the Ion Sequencing Kit User Guide v2.0 (Life Technologies,Part No. 4468997), incorporated herein by reference in their entirety.

Example 13

Table 1 presents data obtained from nucleic acid sequencing experimentsand reports the quality of the nucleic acid sequence data using themethods disclosed herein. Quality metrics such as peak signal, 100Q17and AQ17 are presented. Various companion particles were used in thesequencing experiments and are reported under bead description. The beadsize as reported in the Table refers to companion particles with a rangeof 4.8 um to 7.1 um average cross-sectional diameter. The data wasgenerated using Ion Torrent 316™ Chips (Life Technologies, Part No.4462923).

TABLE 1 Run Bead Peak name Bead description size 100Q17 AQ17 signal LEA191 SOLiD EZ bead enricher(dynal) 6 2,845,898 620,601,222 65 HoF 631SOLiD EZ bead enricher(dynal) 6 2,853,418 626,984,623 69 GAR 659Surfactant free blue sulphate 4.8 2,542,949 554,461,510 65 (IDC/molprobes) LEN 661 Surfactant free blue sulphate 4.8 2,872,290 631,110,27965 (IDC/mol probes) KUB 425 Surfactant free blue sulphate 6.3 2,148,266465,699,949 62 (IDC/mol probes) HOF 626 Surfactant free blue sulphate6.3 2,186,206 465,615,985 59 (IDC/mol probes) LEA 188 Surfactant freeyell green sulphate 5.2 2,340,898 507,212,844 70 (IDC/mol probes) HOF628 Surfactant free yell green sulphate 5.2 2,321,351 502,490,341 69(IDC/mol probes) GAR 660 SPHERO polystyrene blue 7.1 2,801,621621,971,702 68 (Spherotech) LEN 662 SPHERO polystyrene blue 7.12,869,779 630,650,758 68 (Spherotech) LEA 192 SPHERO polystyreneparticles 6.8 2,879,481 632,231,475 69 (Spherotech) HOF 632 SPHEROpolystyrene particles 6.8 3,025,096 669,652,719 69 (Spherotech) GAR 661white nonionic polystyrene 5.2 2,739,361 610,272,589 69 latex(IDC/molprobes) LEN 663 white nonionic polystyrene 5.2 2,456,685 541,576,722 65latex(IDC/mol probes)

Example 14

A sequencing chip can be loaded with a sample following enrichment ofthe sample. The sample may or may not include companion particles.Before starting, the following stock solutions are prepared:

-   -   50% Annealing buffer: in a 15-mL conical tube, combine 5 mL of        Annealing buffer with 5 mL of nuclease-free water.    -   Defoaming solution: in a 15-mL conical tube, combine 5 mL of        100% isopropanol with 2.5 mL of Annealing buffer and 2.5 mL of        nuclease-free water.

Prepare the Chip for Loading

1. Place the Ion Proton I™ Chip (available from ION Torrent) on a stablesurface such as a benchtop.2. In a 1.5 mL tube, combine 64 μL of 50% Annealing buffer with 6 μL offoaming solution.3. Create mock foam by injecting air into the 70 μL sample using aRainin® SR-L200F pipette set to dispense 70 μL. Next, break the largebubbles into smaller bubbles by rapidly pipetting for ˜10 seconds.Repeat this step 2 more times.

-   -   The sample should consist of foam. The foam is ready when it can        be aspirated into the pipette tip as a white, stable mixture of        very small bubbles without aqueous solution collecting at the        tip of the pipette.    -   Be careful not to over-inject air; the final volume of foam        should be approximately 300 μL.        4. Inject 70 μL of mock foam into the chip flow cell. Discard        the liquid that comes out of the opposite port.        5. Proceed immediately to loading the chip.

Load the Sample on the Chip

1. Add 6 μL of Foaming solution to the 64 μL enriched Ion Proton™ I ISPsample and mix, transfer to a 1.5 mL tube.2. Foam up the ISP sample by injecting air into the 70 μL sample using aRainin® SR-L200F pipette set to dispense 70 μL. Next, break the largebubbles into smaller bubbles by rapidly pipetting for ˜10 seconds.Repeat this step 2 more times.

-   -   The sample should now consist of foam. The foam is ready when it        can be aspirated into the pipette tip as a white, stable mixture        of very small bubbles without aqueous solution collecting at the        tip of the pipette.    -   Be careful not to over-inject air; the final volume of foam        should be approximately 300 μL.    -   It is convenient to use the same pipette tip throughout this        part of the loading procedure.        3. Aspirate 75 μL of foam into the pipette tip. Using a quick        pipetting motion, inject the foam into the flow cell. Discard        the mock foam that comes out of the opposite port.        4. Insert the chip into the chip-holding adapter and vortex at        3000 rpm for 2 minutes.        If using an IKA MS3 vortexer, the machine should be in ‘mode B’        to achieve 3000 rpm. To go into ‘mode B’, hold down the Start        button while pressing the Power button when turning on the        vortexer. Vortex Genie vortexers should be run at the maximum        setting.        5. Set the pipette to 65 μL. Pipette the foamy ISP solution up        and down rapidly to make sure the foam is the right consistency.        It is desirable to ‘re-foam’ in this manner before every foam        injection.        6. Inject 65 μL of foam into the flow cell. Save the used foam        that comes out of the opposite port by pipetting it into a 1.5        mL tube labeled ‘used foam’.        7. Vortex the chip for 2 minutes.        8. Repeat steps 5-7 one or two more times until all or most of        the foam has been used.        9. After the last foam injection, if there is a small amount of        foam left in the 1.5 mL tube, transfer it to the ‘used foam’        tube.        10. Pipette the ‘used foam’ up and down rapidly for ˜10 seconds        to ensure that the foam is the right consistency.        11. Inject 65 μL of foam from the ‘used foam’ tube into the flow        cell. Add the foam that comes out of the opposite port to the        ‘used foam’ tube.        12. Vortex the chip for 2 minutes.        13. Repeat steps 11-12 two or three more times until all or most        of the foam has been used. For these injections, discard the        foam that comes out of the opposite port.

Flush Out the Foam and Load Polymerase

1. Flush the chip with 100 μL of a Defoaming solution. Discard thesolution that comes out of the opposite port.2. Some bubbles will remain along the edges of the flow cell. Removethese by slowly withdrawing ˜75% of the defoaming solution from the flowcell, then slowly pipetting it back in.

-   -   It is easiest to perform this step by dialing the pipette and        holding the chip at an angle (rather than flat on the bench)        such that the force of gravity pulls the solution towards the        pipetting port.    -   Pay attention to the bubbles along the edge of the flow cell.        The defoaming solution should disrupt these bubbles as it is        withdrawn from the flow cell.    -   When pipetting the defoaming solution back in, the fluid        meniscus should ‘hug’ the edge of the flow cell without allowing        air pockets to form.    -   The defoaming solution may not completely fill the flow cell        once it has been dialed back in. The flushing procedure in the        next step will usually remove any air pockets that remain.        3. Flush the chip 4 times with 100 μL of 50% Annealing buffer.        Be careful not to introduce air bubbles. After each flushing        step, remove the liquid that comes out of the opposite port.        4. Combine 6 μL of Ion Proton™ I Sequencing 200 Polymerase with        65 μL of 50% Annealing buffer.        5. Slowly inject this solution into the flow cell (this step can        be performed by dialing the pipette).        6. After 5 minutes, clamp the chip into the Ion Proton™        Sequencer and proceed with starting the run.

Example 15

A sequencing chip can be loaded with a sample following enrichment ofthe sample. The sample may or may not include companion particles.Following formation of the enriched sample, a chip can be loaded usingcentrifugation.

1. With the chip on a flat surface, dial in 304 of sample into theloading port (large port) of the chip, making sure that no air bubblesare introduced. Remove residual liquid from outlet.2. Centrifuge the chip for 30 seconds with the tab facing in.3. Mix the sample in the chip: Tilt the chip 45 degrees so that theloading port is the lower port. Set the pipette to ˜30 ul and pipettethe liquid in and out 3 times. Choose a pipetting rate so that most ofliquid is being moved but no air bubbles are introduced into the chip.4. Centrifuge for 30 seconds, with the tab facing out.5. Repeat step 3, followed by a final 30 second spin with the tab in.6. Remove the liquid from the chip: Holding the chip at a 45-degreeangle, gently remove as much liquid as possible from the port by dialingthe liquid out. If all the liquid does not come out, put the chip in thecentrifuge with the tab facing out, and perform a quick ˜5 sec spin. Ifthe liquid is not quite in the port area, firmly tap the chip againstthe table a few times with the point of impact being the tab. Thisshould bring the liquid to the loading port area for pipette removal. Donot flush the chip.

7. Start the PGM run. Example 16

A sequencing chip can be loaded with a sample following enrichment ofthe sample. The sample may or may not include companion particles.

1. After washing 30M enriched beads in PBST, decant the sample untilapproximately 15 uL are remaining. To this, add 12 uL of SequencingPrimer and place tube in thermal cycler. Run ‘FastHyb’ program.2. After chip check and calibration, flush the chip with 200 uL of 100%isopropyl alcohol (surfynol) followed by 200 uL of 50% PBST. Repeat the50% PBST flushes for a total of 3 flushes.3. Take sample tube off of thermal cycler and add 3 uL of sequencingpolymerase. Mix well and allow the sample to incubate for 5 minutes atroom temperature.4. After incubation, add 30 uL of 50% PBST to the sample for a finalvolume of 60 uL.5. Remove liquid from the flowcell.6. Dial 30 uL of the diluted sample into the chip.7. Spin for 1 minute in a (Galaxy) centrifuge.8. Remove chip from centrifuge and add (dial in) the remaining 30 uL ofsample. Collect the ‘flow-through’ (the best way to do this is to dialup the liquid) that exits the chip and inject this into the chip again.Collect the flow-through once more and set it aside.9. Spin for one minute.10. Repeat steps 7-8 four more times (for a total of five ‘flow-through’steps (2×30 uL each)).11. After the final spin, the chip is ready to be loaded on the PGM. Noflushing or backfilling is utilized.

Notes:

1. Injections into the chip should be dialed in. This should not takelonger than 10 seconds per 30 uL.2. Collect the waste from the reservoir through dialing up (to avoidbubbles).3. The volume of the waste decreases slowly over time as it is difficultto collect every last microlitre after each flow. Do not top this upwith 50% PBST, simply continue to flow through whatever volume iscollected.

Example 17

A sequencing chip can be loaded with a sample following enrichment ofthe sample. The sample may or may not include companion particles.

1. Obtain a new 318 chip, label appropriately to identify theexperiment.2. Perform chip check and calibration on the PGM.3. Remove the chip and wash with 100 μL 100% Isopropanol (2-propanol)and then wash the chip 3× with 100 μL Annealing Buffer.

Note: When your chip is not in the PGM and clamped down, add a dummychip to prevent air pockets from forming due to back flow in the squidlines.

Post-Enrichment: Isolate 20 Million SNAPPs (318)

1. Transfer SNAPPS into a 200 μL PCR tube and add 5 μL TFs. If SNAPPsare in more than 50 μL, add Annealing Buffer up to ˜150 μL and spin onceto concentrate, then remove supernatant down to 25 μl.2. Wash SNAPPS by filling the tube with 150 μL Annealing Buffer and tipmix.3. Centrifuge 2 minutes @ a minimum of 15000 rcf and leave 15 μL in thetube. Mix well by pipetting.4. Add 12 μL Sequencing Primer. Confirm volume is 27 μL; if less, addAnnealing Buffer until the final volume is 27 μL. Mix well by pipetting.5. Run the QuickHyb Program on the cycler (95° C., 2 min; 37° C., 2min).6. Add 3 μL of Sequencing Polymerase, tip mixing well. Incubate at roomtemp for 5 minutes. The final volume should be about 30 ul.7. Centrifuge chip upside-down, with tab facing out for 30 seconds toempty buffer from chip (using UP adapter placed over chip). Wipe offexcess buffer on UP adapter with Kimwipe.8. Place the chip on a flat surface, dial in 304 of sample into theloading port (large port) of the chip.9. Centrifuge your chip for 1 minute with the tab facing in.10. Place the adapter onto the chip and centrifuge the chip upside-downwith tab facing out for 1 min.11. Remove the chip from the adapter and pipette the liquid from theadapter well and reload into the chip. (Note: Add in 50% annealingbuffer to fill in any open space in the chip)12. Centrifuge the chip right side up for 1 min with the tab facing in.13. Repeat step 1014. Place the empty chip on the PGM and start the run.

Example 18

A sequencing chip can be loaded with a sample following enrichment ofthe sample. The sample may or may not include companion particles.

Post-Enrichment

1. Transfer SNAPPS into a 200 μL PCR tube and add 5 μL TFs. If SNAPPsare in more than 50 μL, add Annealing Buffer up to ˜200 μL and spin onceto concentrate, and then remove supernatant down to 25 μl.2. Wash SNAPPS by filling the tube with (150-200 μL) Annealing Bufferand tip mix.3. Centrifuge 2 minutes @a minimum of 15000 rcf and leave 15 μL in thetube. Mix well by pipetting.4. Add 12 μL Sequencing Primer. Confirm volume is 27 μL; if less, addAnnealing Buffer until the final volume is 27 μL. Mix well by pipetting.5. Run the QuickHyb Program on the cycler (95° C., 2 min; 37° C., 2min).6. Add 3 μL of Sequencing Polymerase, tip mixing well. Incubate at roomtemp for 5 minutes. The final volume should be about 30 ul. A slightlyhigher volume (up to 35 uL) is better than a lower volume. If lower,adjust the volume with annealing buffer as is useful.7. With the chip on a flat surface, dial in 304 of sample into theloading port (large port) of the chip, making sure that no air bubblesare entrapped. Remove residual liquid from outlet.8. Centrifuge your chip for 1 minute with the tab facing in.9. Leaving the chip in the bucket, add the weighted adapter to the topof the chip. Add 100 ul of 50% annealing buffer to the port areaopposite the weighted side.10. Place the chip into the centrifuge. Make sure the weighted side isfacing the outer edge of the centrifuge before spinning. Centrifuge for30 seconds.11. Lift the adapter off the chip surface, rotate 180 degrees, and placeback down on top of the chip. Once again, make sure the weighted side isfacing the outer edge of the centrifuge before spinning. This amounts tothe chip rotating before each spin, but the adapter staying in the sameorientation. Centrifuge for 30 seconds.12. Repeat step 11 three more times.13. Remove the adapter from the top of the chip and remove the excessliquid from the port area.14. Perform a final 1 minute spin with the tab facing in.

15. Start the PGM run.

In a first aspect, a method for analyzing a biomolecule includes forminga foam from a sample including a plurality of biomolecule-enhancedparticles, applying at least a portion of the foam to an array, andvortexing the array.

In an example of the first aspect, forming the foam includes injectingair into the sample. For example, forming the foam can includeaspirating the foam into a pipette tip repeatedly.

In another example of the first aspect and the above examples, themethod further includes applying another portion of the foam to thearray. In an example, the method further includes vortexing the arrayafter applying the another portion of the foam. In an additionalexample, the method further includes repeating applying another portionof the foam and vortexing.

In a further example of the first aspect and the above examples, themethod further includes removing used foam following vortexing. In anexample, the method further includes placing the array in a device. Themethod can further include analyzing the biomolecules on thebiomolecule-enhanced particles.

In a second aspect, a method of analyzing a biomolecule includesapplying at least a portion of a sample to an array and centrifuging thearray and repeating applying and centrifuging.

In an example of the second aspect, applying includes pipetting the atleast a portion of the sample into a flow cell formed over the array.

In another example of the second aspect and the above examples,repeating includes withdrawing and reapplying the at least a portion ofthe sample to the array.

In a further example of the second aspect and the above examples,repeating includes applying another portion of the sample to the array.

In an additional example of the second aspect and the above examples,applying includes pipetting an amount of the sample that is not greaterthan the volume of a flow cell formed over the array.

In an example of the second aspect and the above examples, applyingincludes pipetting an amount of the sample that is greater than thevolume of a flow cell formed over the array and collecting apass-through portion of the sample.

In another example of the second aspect and the above examples,centrifuging includes centrifuging with the array facing a center pointof the centrifuge.

In a further example of the second aspect and the above examples,centrifuging includes centrifuging with the array facing away from acenter point of the centrifuge.

In an additional example of the second aspect and the above examples,centrifuging includes centrifuging with the array perpendicular to aplane of rotation.

In an example of the second aspect and the above examples, centrifugingincludes centrifuging with the array tilted relative to a plane ofrotation.

In another example of the second aspect and the above examples, thearray is tilted at an angle not greater than 90°. For example, the arraycan be tilted at an angle of at least 15°.

In a third aspect, a method for depositing a plurality of particles intoa plurality of reaction chambers includes forming a particle mixtureincluding a plurality of sample particles having a first averagediameter and a plurality of companion particles having a second averagediameter, contacting the particle mixture with a surface including aplurality of reaction chambers having entries, wherein the average crosssectional diameter of the entries of the plurality of reaction chambersis greater than the first average diameter but less than the secondaverage diameter.

In an example of the third aspect, the contacting includes depositing asample particle of the particle mixture into a percentage of thereaction chambers. For example, the percentage is increased relative tothe percentage of reaction chambers that are filled by a controlparticle mixture that does not include companion particles.

In another example of the third aspect and the above examples, at leastone of the reaction chambers contains no greater than one sampleparticle.

In a further example of the third aspect and the above examples, thecontacting includes depositing no more than one sample particle in atleast one reaction chamber.

In an additional example of the third aspect and the above examples, thecontacting includes depositing each of at least two sample particlesinto different reaction chambers.

In an example of the third aspect and the above examples, the methodfurther includes separating at least one companion particle from thesurface, optionally without dislodging at least one sample particle fromat least one reaction chamber.

In another example of the third aspect and the above examples, themethod further includes agitating the particle mixture after contactingthe surface.

In a further example of the third aspect and the above examples, thesurface comprises an array. For example, the array can include a beadarray, a slide, a microfluidic array, a nanofluidic array, a chip or asemiconductor based array.

In an additional example of the third aspect and the above examples, atleast one of the reaction chambers comprises a channel, groove, pore,nanopore, well or microwell.

In a further example of the third aspect and the above examples, theparticle mixture comprises about 80% to about 98% by weight sampleparticles.

In an additional example of the third aspect and the above examples, thecompanion particles comprise 15% or less of the total number ofparticles in the particle mixture.

In an example of the third aspect and the above examples, the surfacecomprises about 1 million to about 3 billion reaction chambers.

In another example of the third aspect and the above examples, themethod further includes removing at least some portion of the particlemixture from the surface, optionally without dislodging at least onesample particle from the at least one reaction chamber of the surface.

In a further example of the third aspect and the above examples, atleast one sample particle comprises a nucleic acid molecule attached toat least one bead. For example, the at least one bead can includesilica, glass, coated glass, coated polyacrylamide, acrylamide, nylon,plastic, ceramic, polystyrene, porous silicon, or a combination thereof.In an example, the at least one bead includes a label, dye, magnet ordetectable signal.

In an additional example of the third aspect and the above examples, atleast one companion particle comprises silica, glass, coated glass,coated polyacrylamide, acrylamide, nylon, plastic, ceramic, polystyrene,porous silicon, or a combination thereof.

In an example of the third aspect and the above examples, at least onecompanion particle is inert.

In another example of the third aspect and the above examples, themethod further includes applying a sequencing polymerase or a sequencingprimer to the particle mixture.

In a further example of the third aspect and the above examples, themethod further includes applying a foaming solution to the particlemixture.

In an additional example of the third aspect and the above examples, themethod further includes performing one or more nucleic acid sequencingreactions on the at least one sample particle. In an example, the one ormore one or more nucleic acid sequencing reactions can includesingle-stranded or bi-directional sequencing. In another example, theone or more nucleic acid sequencing reactions can identify at least onemutation in the sample particle.

In a fourth aspect, a sequencing component includes an array of wellsand a plurality of biomolecule-enriched particles disposed in the wellsin a 1:1 relationship, wherein the plurality of biomolecule-enrichedparticles are loaded into the wells using a method of any one of theabove described aspects or associated examples.

In a fifth aspect, a method of analyzing a biomolecule includes applyinga reagent to a sequencing component including an array in which aplurality of biomolecule-enriched particles is disposed, the pluralityof biomolecule-enriched particles loaded into the array using a methodof any one of the above aspects or associated examples and detecting achange in local environments proximate to one or more of the pluralityof biomolecule-enriched particles.

Throughout this application various publications, patents, or patentapplications are referenced. The disclosures of these publications,patents, or patent applications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the described subject matter inany way. All literature and similar materials cited in this application,including but not limited to, patents, patent applications, articles,books, treatises, and internet web pages are expressly incorporated byreference in their entirety for any purpose. When definitions of termsin incorporated references appear to differ from the definitionsprovided in the present teachings, the definition provided in thepresent teachings shall control. Unless defined otherwise, all technicaland scientific terms used herein have the same meaning as is commonlyunderstood by one of ordinary skill in the art to which these inventionsbelong. All patents, patent applications, published applications,treatises and other publications referred to herein, both supra andinfra, are incorporated by reference in their entirety. If a definitionor description is set forth herein that is contrary to or otherwiseinconsistent with any definition set forth in the patents, patentapplications, published applications, and other publications that areherein incorporated by reference, the definition or description setforth herein prevails over the definition that is incorporated byreference. It will be appreciated that there is an implied “about” priorto the temperatures, concentrations, times, etc., discussed in thepresent teachings, such that slight and insubstantial deviations arewithin the scope of the present teachings herein.

Note that not all of the activities described above in the generaldescription or the examples are required, that a portion of a specificactivity may not be required, and that one or more further activitiesmay be performed in addition to those described. Still further, theorder in which activities are listed are not necessarily the order inwhich they are performed.

In the foregoing specification, the concepts have been described withreference to specific embodiments. However, one of ordinary skill in theart appreciates that various modifications and changes can be madewithout departing from the scope of the invention as set forth in theclaims below. Accordingly, the specification and figures are to beregarded in an illustrative rather than a restrictive sense, and allsuch modifications are intended to be included within the scope ofinvention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of features is notnecessarily limited only to those features but may include otherfeatures not expressly listed or inherent to such process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive-or and not to an exclusive-or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Also, the use of “a” or “an” are employed to describe elements andcomponents described herein. This is done merely for convenience and togive a general sense of the scope of the invention. This descriptionshould be read to include one or at least one and the singular alsoincludes the plural unless it is obvious that it is meant otherwise.

Benefits, other advantages, and solutions to problems have beendescribed above with regard to specific embodiments. However, thebenefits, advantages, solutions to problems, and any feature(s) that maycause any benefit, advantage, or solution to occur or become morepronounced are not to be construed as a critical, required, or essentialfeature of any or all the claims.

After reading the specification, skilled artisans will appreciate thatcertain features are, for clarity, described herein in the context ofseparate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination. Further, references to valuesstated in ranges include each and every value within that range.

What is claimed:
 1. A method of analyzing a nucleic acid, the methodcomprising: applying at least a portion of a sample solution to anarray, the sample solution comprising a particle substrate coupled to aplurality of copies of the nucleic acid; centrifuging the array while inthe presence of the at least a portion of the sample, the arrayincluding a plurality of wells in cooperative association with aplurality of sensors, the array disposed at an angle relative to aradial vector in a rang eof 0° to 90° during centrifuging, the particlesubstrate depositing into a well of the plurality of wells; andrepeating applying and centrifuging.
 2. The method of claim 1, whereinapplying includes pipetting the at least a portion of the sample into aflow cell formed over the array.
 3. The method of claim 1, whereinrepeating includes withdrawing and reapplying the at least a portion ofthe sample to the array.
 4. The method of claim 1, wherein repeatingincludes applying another portion of the sample to the array.
 5. Themethod of claim 1, wherein applying includes pipetting an amount of thesample that is not greater than the volume of a flow cell formed overthe array.
 6. The method of claim 1, wherein applying includes pipettingan amount of the sample that is greater than the volume of a flow cellformed over the array and collecting a pass-through portion of thesample.